P.1

1974 (including covering contaminated soil with ofthe lead associated with oat tops was directly at­ clean soil and reducing smelter emissions) and tributable to automobile (aerosol) emissions, but it brought about a decrease in blood lead levels that did not distinguish between lead in the edible por­ were measured a year later. The details ofthe blood tion (grain) and lead on the hulls or chaff.27 This lead levels and their significance are presented in same study reported that lettuce grown in the Salinas Chapter 12. The conclusion to be drawn from this Valley had 3 to 25 ppm lead (dry weight) associated study (and from the similar studies referred to with it, whereas the soil lead content was only 10 above) is that people who live in the vicinity ofa ma­ ppm. The lead content in the lettuce was reported to jor industrial source of lead (e.g., a smelter) are ex­ be 0.15 to 1.5 ppm on a fresh weight basis. The posed to abnormally high lead concentrations. limited data accumulated were used to deduce that the excess lead was delivered to the lettuce by aerial 7.4 DIETARY EXPOSURES emission from autos, and that removal of lead from 7.4.1 Food automobile exhaust would reduce the lead content 27 The route by which most people receive the of the lettuce by as much as 80 percent. In other largest portion of their daily lead intake is through areas, the contribution would be smaller. Though foods, with estimates ofthe daily dietary lead intake these figures may be accurate, they are based on for adult males ranging from 100 to 500 /Ag/day.26 some rather tenuous assumptions that are not well Only a fraction of this ingested lead is absorbed, as supported by observation and very limited data. The discussed in Chapter 10. estimates must thus be considered with caution. The sources of the lead content of unprocessed Moreover, one cannot extrapolate from lettuce or vegetable foods have been noted earlier (Section oats to all crops. 6.4.3). Studies of the lead associated with crops The possible connection between air lead and (near highways) have shown that both lead taken up food lead may be underscored by comparisons be­ from soil and aerosol lead delivered by deposition tween leafy vegetables and food grains. Studies have are found with the edible portions of common shown that the edible portions of grains absorb very vegetable crops. However, there is enormous little air lead,32 whereas the leafy vegetables retain variability in the amount of lead associated with appreciable quantities.2729 An FDA survey34 shows such crops and in the relative amounts of lead in and that grains contain approximately 20 percent as on the plants. Several factors are involved, the most much lead as the leafy vegetables. It cannot be con­ prominent of which are: the plant species, the traffic cluded, however, that 80 percent of the lead in all density, the meteorological conditions, and the local leafy vegetables derives directly from air because soil conditions.2733 The variability induced by the difference must also reflect species-dependent differences in the above factors, coupled with the differences in uptake from soil. fact that many studies have neglected differentiation An overall analysis ofthe data available supports between lead on plants versus lead in the plants, the contention that plants grown near busy highways makes it difficult to generalize. Data of Schuck and consistently have more lead in and on them than 29 Locke suggest that in some cases (e.g., tomatoes those in other areas. This difference is typically very and oranges), much of the surface lead is readily hard to detect at distances greater than about 100 to removed by washing. But as noted in Section 6.4.3, 200 m from the highway, thereby reflecting the fact this is not universally true; in some cases much more that large percentages of the aerosol lead fall out vigorous washing procedures are required. near roadways. It appears reasonable to point out In view of the wide variability of soil conditions that the vast majority of edible crops marketed in (pH, organic matter, cation exchange capacity, this country are grown at distances of more than 100 phosphorus content, etc.), of meteorology to 200 m from the highway and that much of the (especially wind conditions and rainfall), and ofthe aerosol lead can be removed from association with effects of species diversity on the routes of lead ac­ the plants by processing. Clearly there are excep­ cumulation, only crude general correlations between tions that will influence both sides of the question. air lead levels and food crop lead levels are possible. For the present, however, the available data are not This is influenced by the fact that the lead associated sufficient to permit the quantitative estimate of the with plants may be derived from natural sources, contribution of automotive lead to foodstuffs on a from automotive sources, and from other sources national or even regional scale. such as manufacturing or combustion. One study in The concentrations of lead in various food items Southern California reported that 60 to 70 percent are highly variable, and as much variation is found

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within specific food items as between different food Canned pet foods have been found to contain 0.9 categories. Schroeder and Balassa,35 in a study of to 7.0 ppm lead (approximately 900 to 7,000 American foods, have found that the ranges are 0 to /xg/liter),40 and 18 products averaged 2.7 ppm (ap­ 1.5 mg/kg (ppm) for condiments, 0.2 to 2.5 mg/kg proximately 2700 /xg/liter). Apart from the possible for fish and other seafood, 0 to 3.7 mg/kg for meats toxic effects on pets, the products pose a hazard to and eggs, 0 to 1.39 mg/kg for grains, and 0 to 1.3 persons who may include them in their own diet. mg/kg for vegetables. All of these values refer to The lead content in milk is of special interest unprocessed foods. A British report36 on lead in because it is a major component of the diets of in­ foods describes similar ranges for meat and eggs, fants and young children. The FDA survey37 found grain products (flour and bread), and vegetables; lead concentrations in whole milk ranging from 10 but concentrations up to 14 mg/kg were found in to 70 tig/liter and averaging about 20 pig/liter. In a condiments, and up to 18 mg/kg in certain shellfish. recent study by Ziegler et al.,41 seven samples of The amount of lead taken in with food varies from baby formula and three samples of whole cow's milk person to person. It depends on (a) the total amount were analyzed in duplicate. Mean concentrations of of food eaten, (b) the history of the food during lead were 18 /xg/kg (range 15 to 20) in formula and growth, (c) its opportunity to acquire intrinsic lead 10 /xg/kg (range 7 to 15) in milk (1 /xg/kg is approx­ (absorbed from soil or water) and extrinsic lead imately 1 /xg/liter). Lead concentration in infant (deposited insecticides or contaminated dusts), and fruit juices ranged from 23 to 327 /xg/kg; in five (d) dietary habits (such as using fresh rather than varieties of strained fruits, it ranged from 13 to 131 canned foods). On a per-weight basis, the dietary in­ /xg/kg; and in seven varieties of strained vegetables, take oflead by children has been shown to be two or lead concentration was 14 to 73 /xg/kg. Tolan and 38 three times that of adults. This additional dietary in­ Elton reported 30 /xg Pb/liter in fresh milk in take is especially significant when the lead added to Great Britain and 50 /xg Pb/liter in canned (evapor­ 39 food by processing and to water by plumbing (vide ated) milk. Michell and Aldous reported a com­ infra) is considered. A 1974 FDA survey of heavy parable average for fresh whole milk purchased in metals in foods37 found relatively high lead con­ New York State —40 /xg Pb/liter. But their results centrations in metal-canned foods. In the adult food for evaporated milk averaged 202 /xg Pb/liter and category, canned foods averaged 0.376 ppm lead, ranged as high as 820 /xg Pb/liter. and non-canned foods averaged 0.156 ppm lead. In Hankin et al.42 suggest an additional food-related the baby food category, canned foods (juices) source of potential lead exposure, again predomi­ averaged 0.329 ppm lead, and foods in jars averaged nantly affecting children. The colored portions of 0.090 ppm. The report of the survey concludes that wrappers from bakery confections, candies, gums, from the age of about 1 year on, canned foods com­ and frozen confections have lead concentrations prise 11 to 12 percent of a person's diet, but they ranging from 8 to 10,100 ppm. The higher con­ contribute about 30 percent of the average dietary centrations are attributed to lead-containing inks. lead intake. In a comparison made in the United 38 No related illnesses were identified, nor was con­ Kingdom, lead concentrations in canned foods tamination of the food implied; but the eating of were found to vary widely with the precise nature of foods from such wrappers and the licking or chewing the food, but they averaged about ten times greater ofthe wrappers were postulated as one more avenue than those in fresh foods. for an additional increment to total lead exposure. The soldered seam of tin cans is evidently the ma­ The presence of high lead concentrations in illicit jor source of this additional lead in canned foods, whiskey (moonshine), which is still popular in some and increasing lead concentrations in samples of a parts of the United States despite the repeal of can's contents taken progressively nearer the seam prohibition, causes lead poisoning in adults. The ap­ have been found.39 Similarly, there is a correlation parent source of the lead is the soldered joints in the between increasing lead concentrations in canned distilling apparatus. products and the increasing ratio of the can's seam Another potential source of dietary lead poisoning length to volume. Of 256 metal-canned foods ex­ is the use of inadequately glazed earthenware vessels amined, 37 percent contained 200 /xg Pb/liter or for food storage and cooking. An impressive exam­ more; 12 percent contained 400 /xg Pb/liter or more. ple of this danger involved the severe poisoning of a These levels are markedly above the potable water physician's family in Idaho and stemmed from standard of 50 tig Pb/liter (0.05 mg/kg) established drinking orange juice that had been stored in an by the U.S. Public Health Service. earthenware pitcher.43 Similar cases, sometimes in-

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eluding fatalities, have involved other relatively lem. The extensive use of lead compounds as acidic beverages such as fruit juices and soft drinks gasoline additives has greatly increased the and have been documented by other workers.4445 availability of lead for solution in ground and sur­ 46 47 face waters. For example, in a study in east-central Recent reports on lead in European wines - 51 show concentrations typically averaging from 130 to Illinois, the urban portion of an 86-square-mile 190 /xg/liter (0.13 to 0.19 ppm) and ranging as high watershed, which constituted 14 percent ofthe area, as 299 /xg/liter (0.299 ppm). Measurements of lead contributed about 75 percent of the lead in the in domestic wines have not been undertaken; but if drainage waters. The principal source of this lead is the European data are indicative, wines could con­ identified as automotive emissions. Detailed data re­ ported for 1 month (June 1972) show that drainage tain lead concentrations comparable to processed waters from this urban portion contained an average foods previously discussed. total lead concentration of 69.5 /xg/liter, including 7.4.2 Water 6.3 /xg/liter of soluble lead. The rural portion yielded an average of lead concentration of 7.4 The U.S. Public Health Service's standards for /xg/liter of drainage water, including 2.1 /xg/liter of drinking water specify that lead should not exceed soluble lead. 50 /xg/liter (0.05 ppm). The average adult drinks about 1 liter of water per day. The presence of detec­ The major source of lead contamination of drink­ table amounts of lead in untreated public water sup­ ing water is the water supply system itself. Water plies was shown by Durum48 to be widespread, but that is corrosive can leach considerable amounts of only a few samples contained amounts above the 50 lead from lead plumbing and lead compounds used /xg/liter standard. Durfor and Becker49 analyzed to join pipe. Several widely adopted codes, such as untreated and treated water for the largest U.S. the ASA-A40 Code, Uniform Plumbing Code, and cities, and almost all pairs of samples showed a sub­ BOCA Code, allow the use of lead pipe and list lead stantial decrease in lead that was ascribable to treat­ as an acceptable soldering material for joining pipes ment provided. A maximum lead concentration of that convey water. Lead pipe is currently in use in 62 /xg/liter was detected in finished water from one many parts of the United States for water service of several wells used in Salt Lake City to supplement lines and interior plumbing, particularly in older ur­ their surface water supply. Some 95 percent of the ban areas. In a community water supply survey of water supplies sampled, however, had less lead than 969 water systems conducted in nine geographically 10 /xg/liter in the treated water before entering the distributed areas of the United States in 1969 and distribution system. Eight of the water supplies dis­ 1970, it was found that 1.4 percent of all tap water tributed water with a pH of less than 7, which could samples exceeded the 50 /xg/liter standard.52 The be corrosive to the distribution piping; most ofthese maximum concentration found was 640 /xg/liter were in the Northwest. A chemical analysis of 592 total lead. The occurrence of samples exceeding the interstate carrier water supplies in 1975 showed only standard was more prevalent in waters with a 0.3 percent to exceed the 50 /xg/liter standard.50 relatively low pH and low specific conductance. It These samples were collected after treatment but was estimated that 2 percent of the survey popula­ before distribution, and they represent both sus­ tion of 18.2 million was exposed to high lead levels pended and dissolved lead. Interstate carrier water at the tap. supplies serve planes, trains, buses, and vessels in in­ Hem and Durum53 discuss the solubility of those terstate commerce, and they include almost all of species of lead that may be present in drinking water the largest U.S. water supplies. and suggest that the solution of lead from environ­ The presence of lead in drinking water may result mental sources may be an important contribution in from contamination ofthe water source or from the certain areas, depending on the chemical composi­ use of lead materials in the water distribution tion ofthe runoff water. Above pH 8.0, the solubility system. Although lead is a relatively minor constit­ of lead is below 10 /xg/liter, regardless of the uent of the earth's crust, it is widely distributed in alkalinity of the water. In waters near pH 6.5 with a low concentrations in sedimentary rock and soils (as low alkalinity, however, the solubility of lead could discussed in Chapter 3), and naturally occurring approach or exceed 100 /xg/liter. Lazrus et al.54 deposits may be an important source of contamina­ determined the lead content of precipitation at 32 tion in isolated instances. Industrial waste may also points in the United States for a period of 6 months contribute to the lead content of water sources, but in 1966 and 1967. They reported an average lead this appears to be a local and not a widespread prob­ concentration of 34 /xg/liter after filtering the sam-

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pies. Samples of rainfall at Menlo Park, Calif., dur­ areas, the major route of lead exposure is by inhala­ ing 1971 showed a wide range of lead concentra­ tion and ingestion of both lead-bearing dusts and tions, from a few /xg/liter to more than 100 fumes. Airborne dusts settle out from the air onto 53 53 /xg/liter. Hem and Durum hypothesize that high­ food, water, the workers' clothing, and other objects er lead concentrations should be anticipated in and are then transferred to the mouth in one fashion runoff water and impounded raw water supplies in or another. Therefore, good housekeeping and, the Northeast, certain urban areas ofthe South, and above all, good ventilation have a strong impact on along the Pacific Coast because of low pH and exposure. Exposure levels have been found to be alkalinity in waters. However, in much ofthe rest of quite high in one factory and quite low in another the United States, lead fallout rates and the chemical solely because of differences in ventilation engineer­ composition of the runoft (pH >8; alkalinity > 100 ing or housekeeping practices and worker education. mg/liter) would minimize the problem. Information to test their hypothesis is limited at present. Of the 7.5.1 Exposures in Lead Mining, Smelting, and few surveys of surface waters that have been con­ Refining ducted, most were not done after periods of heavy The greatest potential for high-level exposure ex­ rainfall, and the surveys that have been done have 56 48 ists in the process of lead smelting and refining. measured dissolved rather than total lead. Durum The most hazardous operations are those in which measured lead at 700 lake and river sites in the molten lead and lead alloys are brought to high tem­ United States. These measurements were primarily peratures, resulting in the vaporization of lead. This single samples taken at times of relatively low is because condensed lead vapor or fume has, to a stream flows in October and November 1970. substantial degree, a small (respirable) particle size Detectable concentrations of dissolved lead (>1 range. Thus although the total air lead concentra­ /xg/liter) were found in 63 percent of the samples, tion may be greater in the vicinity of ore-proportion­ but only three samples contained more than 50 ing bins than it is in the vicinity of a blast furnace in a /xg/liter. A large proportion of the samples for the primary smelter, the amount of particle mass in the northeastern and southeastern states contained lead respirable size range may be much greater near the above the detection limit, and quite a few ofthe sam­ furnace. ples showed levels above 10 /xg/liter. This regional distribution ot lead in stream water is in accord with A measure of the potential lead exposure in pri­ mary smelters was obtained in a study of three typi­ the idea that water composition in the eastern states 56 is more commonly favorable for solution of lead. A cal installations in Utah. Air lead concentrations substantial number of samples from southern near all major operations, as determined using per­ sonal monitors worn by the workers, were found to California were high in lead, and these, influenced 3 the data from the southwestern states. Kopp and vary from about 100 to more than 4000 /xg/m . Ob­ Kroner55 presented data on dissolved lead in rivers viously, the hazard to these workers would be ex­ and lakes of the United States. The data were tremely serious were it not for the fact that the use of gathered over a 5-year period (1962 to 1967) and respirators is mandatory in these particular smelters. represent more than 1500 samples. A detectable Although there are no comparable data tor ex­ concentration of dissolved lead was found in 305, or posures in secondary smelters, which are found in or 19.3 percent of the samples; the observed values near most large cities, the nature oftheir operation is ranged from 2 to 140 /xg/liter. The highest con­ similar to that of primary smelters except that no centration was detected on the Ohio River at ore-processing is involved, since secondary smelters Evansville, Indiana. Twenty-seven of their samples depend on the local supply of lead scrap in the form exceeded 50 /xg/liter. Observed mean observations of discarded electric storage batteries, cable casings, of >30 /xg/liter dissolved lead were found in the pipes, and other materials for their supply of lead. following river basins: Ohio, Lake Erie, Upper Consequently, the exposure hazard to workers in Mississippi, Missouri, Lower Mississippi, and Col­ secondary smelters is probably similar to that found orado. in the primary smelter study. Hundreds, perhaps thousands, of the small scrap dealers that supply these secondary smelters have their own neighbor­ 7.5 OCCUPATIONAL EXPOSURES hood or even backyard melting operations for ex­ The highest and most prolonged exposures to lead tracting and reclaiming lead. These operations can are found among workers in the lead smelting, refin­ contribute substantially to local airborne lead ing, and manufacturing industries.56 In the work levels.

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High levels of atmospheric lead are also found in which accumulates as the paste dries. Also the form­ foundries in which molten lead is alloyed with other ing and stacking processes are dusty, and ventilation metals. Berg and Zenz57 found in one such operation is needed there also. The data cited are sufficiently that average concentrations of lead in various work alarming to suggest that respirators must be worn in areas were 280 to 600 /xg/m3. These levels were sub­ most of these operations. sequently reduced to 30 to 40 /xg/m3 with the in­ stallation of forced ventilation systems to exhaust 7.5.4 Exposures in the Printing Industry the work area atmospheres to the outside. In a printing establishment, the exposure to lead is Exposures for workers involved in lead mining probably in direct proportion to the dispersion of depend to some extent on the solubility ofthe lead lead oxide dust, secondary to the remelt operation. from the ores. The lead sulfide (PbS) in galena is in­ Brandt and Reichenbach61 have reported on a 1943 soluble, and absorption through the lung may be study in which melting pots were located in a variety slight. It is not really known how readily absorption of places where used type was discarded. The pots takes place. In the stomach, however, some lead were maintained at temperatures ranging from 268° sulfide may be converted to slightly soluble lead to 446°C. The highest air lead concentration chloride, which may then be absorbed in moderate recorded was 570 /xg/m3. Since this report was amounts. published, working methods and industrial hygiene conditions have changed considerably; but a 7.5.2 Exposures in Welding and Shipbreaking marginal degree of hazard still prevails. In 1960, When metals that contain lead or are protected Tsuchiya and Harashima62 found in several printing with a lead-containing coating (paint or plating) are shops in Japan lead levels of 30 to 360 /xg/m3 at heated in the process of welding or cutting, copious breathing level. quantities of lead particles in the respirable size range are emitted into the air. Under conditions of 7.5.5 Exposures in Alkyl Lead Manufacture poor ventilation, electric arc welding of zinc sili­ Workers involved in the manufacture of both cate-coated steel (determined to contain some 29 mg tetraethyl lead and tetramethyl lead, two alkyl lead Pb/in2 of coating) produced breathing-zone con­ compounds, are exposed to both inorganic and alkyl centrations oflead reaching 15,000 /xg/m3, far in ex­ lead. Some exposure also occurs at the petroleum cess of 450 /xg/m3, the current occupational short- refineries where the two compounds are blended term exposure limit (STEL) in the United States.58 into gasoline, but no exposure data are available on Under good ventilation conditions, a concentration these blenders. of 140/xg/m3 was measured.59 The major potential hazard in the manufacturing In a study of salvage workers using oxy-acetylene of tetraethyl lead and tetramethyl lead is from skin cutting torches on lead-painted structural steel absorption, but this is guarded against by the use of under conditions of good ventilation, breathing- protective clothing. Linch et al.63 found a correla­ zone concentrations of lead averaged 1200 /xg/m3 tion between an index of organic plus inorganic air and ranged as high as 2400 /xg/m3.60 lead concentrations in a plant and the rate of lead excretion in the urine of the workers. The average 7.5.3 Exposures in the Electric Storage Battery In­ concentration of organic lead in the urine was 0.179 dustry mg/m3 for workers in the tetramethyl lead operation At all stages in battery manufacture except for and 0.120 mg/m3 for workers in the tetraethyl lead final assembly and finishing, workers are exposed to operation. The tetramethyl lead reading was proba­ high air lead concentrations, particularly lead oxide bly higher because the reaction between the organic dust. Air lead concentrations as high as 5400 /xg/m3 reagent and lead alloy takes place at a somewhat have been recorded in some studies.56 The hazard in higher temperature and pressure than that employed plate casting, which is a molten-metal operation, is in tetraethyl lead production. from the spillage of dross, resulting in dusty floors. During oxide mixing, which is probably the most 7.5.6 Exposures in Other Occupations hazardous occupation, ventilation is needed when In both the rubber products industry and the the mix is loaded with lead oxide powder, and fre­ plastics industry there are potentially high exposure quent cleanup is necessary to prevent the accumula­ levels to lead. The potential hazard ofthe use of lead tion of dust. In the pasting ofthe plates, whether by stearate as a stabilizer in the manufacture of poly­ hand or machine, the danger again is from dust vinyl chloride was noted in the 1971 Annual Re-

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port of the British Chief Inspector of Factories.64 to be disposed of annually, of which about 0.59 The Inspector stated that the number of reported times IO9 kg would probably be incinerated.70 cases of lead poisoning in the plastics industry was Assuming that lead will be emitted from the un­ second only to that in the lead smelting industry. controlled incineration of PVC's at the rate of 0.2 g Scarlato et al.65 and Maljkovic66 have reported on Pb/kg of waste71 (a figure applying to all solid other individual cases of exposure. The source ofthe waste), about 1.2 times 105 kg of lead could be problem is the dust that is generated when the lead released per year. This would be an increase of more stearate is milled and mixed with the polyvinyl than fourteenfold over the estimate for 1975. Since chloride and the plasticizer. the greater part of the lead in these incinerated Sakurai et al.67 in a study of bioindicators of lead plastic wastes will remain in the ash, electrostatic exposure, found ambient air concentrations averag­ precipitators can substantially decrease the emitted ing 58 /xg/m3 in the lead covering department of a fraction (to an estimated 0.03 g/kg). But this process rubber hose manufacturing plant. Unfortunately, no only aggravates the difficulties of residual solid ambient air measurements were taken for the other waste disposal with its attendant problems of fugi­ departments or the control group. tive dust and the potential contamination of soil, surface waters, and groundwaters through leaching 7.5.7 Exposures Resulting from Manmade from landfill operations. Materials Lead is present in other products that may con­ At least two manmade materials in widespread use stitute sources of lead exposure when used or dis­ are known to contain lead: paint and plastics. posed of. Lead may be found in color newsprint, In 1974, the Consumer Product Safety Commis­ craft and hobby materials, toothpaste tubes, cos­ sion collected selected household paint samples and metic products, candle wicks, pewter and silver 68 analyzed them for lead content. Analysis of 489 hollowware, painted utensils, and decals on samples showed that 8 percent of oil-based paints glassware. For example, lead in the paint on handles and 1 percent of water-based paints contained of kitchen utensils has been found by Hankin et al.72 greater than 0.5 percent lead (5000 /xg Pb/g paint, to range from 0 to 9.7 percent (0 to 97,000 ppm). based on dried solids), which was the statutory limit More than half the paint samples (13 of 21) ex­ at the time of the study. The current statutory limit ceeded the allowable limit for painted toys, which is for Federal construction is 0.06 percent. This limit is 0.06 percent. equivalent to 600 /xg Pb/g paint.68 Old paint that is still on buildings will continue to pose a potential 7.5.8 Historical Changes hazard for some time. It can become accessible Perhaps the most impressive data on the mag­ through flaking, even though painted over with non­ nitude of environmental contamination by lead and toxic paints. its increase over time are to be found in a small Lead in paint constitutes a potential health prob­ group of recent historical studies that examined lem primarily for children with pica who may levels ofthe metal in polar snow and ice (Chapter 6). habitually ingest 1 to 3 g (or more) of paint per Of particular interest was the 200-fold increase in 68 lead levels over several centuries found by week. 73 Plastics contain a number of heavy metals that are Murozumi et al. in the interior of northern Green­ land, and the 10-fold increase through the last cen­ constituents of organometallic stabilizers added 74 during manufacture. The most commonly used lead- tury of ice layers reported by Jaworowski in a study of two Polish glaciers. These findings parallel containing stabilizer is dibasic lead stearate, in 75 amounts ranging from 0.5 to 2.0 parts per 100 parts the results of a study by Ruhling and Tyler, which of resin.69 This stabilizer is normally used in rigid indicated an approximate fourfold increase in lead PVC products. Diffusion, or leaching by solvents, is content in Swedish moss samples taken from the estimated to be quite slow — on the order of 10-'° to period 1890 to the present. These historical records 10-12 cm2/sec at room temperature — but no defini­ reflect the increasing distribution of lead caused by tive information is available. man. Incineration of lead-containing plastics may 7.6 REFERENCES FOR CHAPTER 7 become an increasingly significant source of 1. Akiand. G. G. Air Quality Data for Metals. 1970 through localized lead pollution. It has been estimated that 1974. from the National Air Surveillance Networks. U.S. Environmental Protection Agency. Office of Research and in the year 2000, for example, there could be ap­ Development, Research Triangle Park, N.C. Pub. No. 9 proximately 2.54 times IO kg of PVC plastic waste EPA 600/4-76-041. 1976. 154 p.

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2. Shearer, S. D., G. G. Akiand, D. H. Fair, T. B. McMullen. 16. Ter Haar, G.. and R. Aronow. New information and lead and E. C. Tabor. Concentrations of particulate lead in the in dirt and dust as related to the childhood lead problem. ambient air ofthe United States. Paper presented at Public Environ. Hlth. Persp. 7:83-90, 1974. Hearing on Gasoline Lead Additives Regulations. Los 17. Shapiro. I. M.. H.'L. Needleman, B. Dobkin, and O. C. Angeles. May 2-4, 1972. Tuncay. Lead levels in denture and circumpulpal dentine 3. Faoro, R. B.. and T. B. McMullen. National Trends in of deciduous teeth of normal and lead exposed children. Trace Metals in Ambient Air, 1965-1974. U.S. Environ­ Clin. Chem. Acta. 46(2):\ 19-123. 1973. • mental Protection Agency, Office of Air Quality Planning 18. Position on the Health Implications of Airborne Lead. and Standards, Research Triangle Park, N.C. Pub. No. U.S. Environmental Protection Agency, Office of Health EPA-450/1-77-003. 1977.41 p. and Ecological Effects, Washington, D.C. National Tech­ 4. Motor Gasolines. U.S. Energy Research and Development nical Information Service, Springfield, Va. Pub. No. NTIS Administration, Bartlesville Energy Research Center. PB 228 594/8. 1973. Bartlesville, Oklahoma. 19. Rameau, J. T. Lead as an environmental pollutant. In: 5. Lee. R. E.. S. S. Goranson, R. E. Enrione, and G. B. Proc. Inter. Symp. on Environmental Health Aspects of Morgan. The NASN cascade impactor network. Part 11. Lead. Amsterdam, October 1972. Luxembourg, Commis­ Size distribution measurements of trace-metal compo­ sion ofthe European Communities. 1973. p. 189-200. nents. Environ. Sci. Tech. 6(1 2):1025-1030, 1972. 20. Lead: Airborne Lead in Perspective. National Academy of 6. Darrow, D. K. and H. A. Schroeder. Childhood exposure Sciences, Washington, D.C. 1972. p. 29. to environmental lead. Presented at American Chemical 21. Needleman, H. L. and J. Scanlon. Getting the lead out. Society National Meeting, Chicago, III.. August 29. 1973. New Eng. J. Med. 288(9):466-467, 1973. 7. Edwards, H. W. Environmental contamination by 22. Landrigan, P. J., S. H. Gehlbach, B. F. Rosenblum, J. M. automotive lead. In: Inter. Symp. Proc. on Recent Ad­ Shoults, R. M. Candelaria, W. F. Barthel, J. A. Liddle. A. vances in the Assessment ofthe Health Effects of Environ­ L. Smrek. N. W. Staehling, and J. D. F. Sanders. Epidemic mental Pollution, Vol. III. Paris. June 1974. Luxembourg. lead absorption near an ore smelter: The role of particu­ Commission of the European Communities. 1975. p. late lead. New Eng. J. Med. 292(3):1 23-129, 1975. 1277-1286. 23. Needleman. H. L.. I. Davidson, E. M. Sewell. and I. M. 8. Barltrop, D.. and C. D. Strelow. Westway Nursery Testing Shapiro. Subclinical lead exposure in Philadelphia school Project. Report to the Greater London Council. August children: Identification by dentine lead analysis. New Eng. 1976. J. Med. 290(5):245-248, 1974. 9. PedCo-Environmental, Inc. Lead Analysis for Kansas City 24. Yankel, A. J.. I. H. von Lindern. and S. P. Walter. The and Cincinnati. Report to U.S. Environmental Protection Silver Valley lead study: The relationship between child­ Agency. Research Tri-Park. N.C. PN3264-E. March 1977. hood blood lead levels and environmental exposure. J. Air 10. Second Annual Catalyst Research Program Report: Sup­ Poll. Contr. Assoc. 27(8):763-767. 1977. plement II. U.S. Environmental Protection Agency, Office 25. Landrigan, P. J.. E. L. Baker, R. G. Feldman, D. H. Cox, of Research and Development. Research Triangle Park. K. V.Eden, W. A. Orenstein, J. A. Mather, A. J. Yankel, N.C. 1977. p. 359-466. and I. H. von Lindern. Increased lead absorption with I 1. Harrison, R. M.. R. Perry, and D. H. Slater. The contribu­ anemia and slowed nerve conduction in children near a tion of organic lead compounds to total lead levels in ur­ lead smelter. J. Pediatrics. 89(6):904-9l0. Dec. 1976. ban atmospheres. In: Proc. Inter. Symp. on Recent Ad­ 26. Schroeder, H. A. and I. H. Tipton. The human body vances in the Assessment ofthe Health Effects of Environ­ burden oflead. Arch. Environ. Health 7 7:965-978, 1968. mental Pollution, Vol. III. Paris, June 24-28. 1974. Lux­ 27. Rabinowitz. M. B. Lead Contamination ofthe Biosphere embourg, Commission of the European Communities. by Human Activity. Ph.D. Dissertation, University of 1975. p. 1783-1788. California at Los Angeles. 1974. 12. Fritsch, A. and M. Prival. Response to the United States 28. Motto, H. L„ R. H. Daines, D. H. Chilko, and C. K. Mot­ Environmental Protection Agency's notice for additional to. Lead in soils and plants: Its relationship to traffic health effects information concerning the use of leaded volume and proximity to highways. Environ. Sci. Technol. gasoline. Center for Science in the Public Interest. Wash­ 4(3):231-238, 1970. ington, D.C. 1972. 11 p. 29. Schuck, E. A. and J. K. Locke. Relationship of automotive 1 3. Kennedy, G. L., Jr. Blood and Tissue Lead Content Study lead particulates to certain consumer crops. Environ. Sci. Following Exposure of Male Albino Rats to Lead-Con­ Technol. 4(4):324-330, 1970. taining Expressway Dirt, Neighborhood Street Dirt, Lead 30. Welch, W. R. and D. L. Dick. Lead concentrations in Acetate, or Lead Oxide. Industrial Bio-Test Laboratories. tissues of roadside mice. Environ. Pollut. 8:15-21, 1975. Northbrook, Illinois. Research Report IBT No. E1733C. 31. Dedolph, R..G. Ter Haar, R. Holtzman, and H. Lucas, Jr. 1973. 16 p. Sources of lead in perennial ryegrass and radishes. En­ 14. Lombardo, L. V. The Public Interest Campaign, Washing­ viron. Sci. Technol. 4(3):217-223, 1970. ton, D.C. Personal Communication to Mr. Wm. D. 32. Ter Haar, G. Air as a source of lead in edible crops. En­ Ruckelshaus, Administrator, United States Environmental viron. Sci. Technol. 4(3):226-229, 1970. Protection Agency. March 9, 1973. 33. Arvik, J. H. Factors Affecting Uptake of Lead by Plants. 15. Pinkerton.C.J. P. Creason. D. I. Hammer, and A. V. Col­ Ph.D. Dissertation, Colorado State Univ., Fort Collins, ucci. Multimedia Indices of Environmental Trace Metal Colo. 1973. Exposure in Humans. In: Proc. 2nd Inter. Symp. on Trace- 34. Kolbye, A. C. K. R. Mahaffey, J. A. Fiorino, P. C. Cor­ Element Metabolism in Animals, Vol. 2. Madison, Wis. neliussen, and C. F. Jelinek. Food exposure to lead. En­ 1973. p. 465-469. viron. Health Persp. 7:65-74, 1974.

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35. Schroeder, H. A. and J. J. Balassa. Abnormal trace metals 55. Kopp, J. F. and R. C. Kroner. Trace Metals in Waters of in man: Lead. J. Chron. Dis. /4(4):408-425, 1961. the United States; A Five-Year Summary of Trace Metals 36. Survey of Lead in Food. (Second Report). Appendix IV. in Rivers and Lakes ofthe Untied States (Oct. 1, 1962 - Ministry of Agriculture, Fisheries and Food. Her Ma­ Sept. 30, 1967). U.S. Dept. of Interior, Federal Water jesty's Stationery Office, London. 1972. Pollution Control Administration, Cincinnati, Ohio. 37. Compliance Program Evaluation, FY-1974, Heavy Metals 1967. 218 p. in Foods Survey. U.S. Food and Drug Administration, 56. Environmental Health Criteria. Vol. 3., Lead. United Na­ Bureau of Foods. Washington. D.C. 1975. 99 p. tions Environment Program/World Health Organization, 38. Tolan, A., and G. A. H. Elton. Lead intake from food. In: Geneva. Switzerland. 1977. p. 59-65. Proc. Inter. Symp. on Environmental Health Aspects of 57. Berg, B. A., and C. Zenz. Environmental and clinical con­ Lead. Amsterdam, October 1972. Commission of the trol of lead exposure in a nonferrous foundry. J. Amer. European Communities, Luxembourg. 1973. p. 77-84. Ind. Hyg. Assoc. 28(2): 175-178, 1967. 39. Michell, D. G. and K. M. Aldous. Lead content of 58. Pegues. W. L. Lead fume from welding on galvanized and foodstuffs. Env. Hlth. Pers. 7:59-64, May 1974. zinc-silicate coated steels. J. Amer. Ind. Hyg. Assoc. 40. Hankin, L.. G. H. Heichel, and R. A. Botsford. Lead con­ 2/(3):252-255,1960. tent of pet foods. Bull. Environ. Contamin. and Toxicol. 59. Tabershaw, I. R.. B. P. W. Ruotolo, and R. P. Gleason. /5(5):630-632. 1975. Plumbism resulting from oxyacetylene cutting of painted 41. Ziegler. E. E., B. B. Edwards, R. L. Jensen. K. R. structural steel. J. Ind. Hyg. Toxicol. 25(5): 189-191. Mahaffey, and S. D. Fomon. Absorption and retention of 1943. lead by infants. (In press.) Pediatric Research. 60. Rieke, F. E. Lead intoxication in shipbuilding and 42. Hankin. L.. G. H. Heichel, and R. A. Botsford. Lead on shipscrapping, 1941-1968. Arch. Env. Health. wrappers of specialty foods as a potential hazard for 79:521-539, 1969. children. Clinical Pediatrics. /5(12):1064-I065. 1974. 61. Brandt. A. D. and G. S. Reichenbach. Lead exposures at 43. Block, J. L. The accident that saved five lives. Good the government printing office. J. Ind. Hyg. Toxicol. Housekeeping, November 1969. 25( 10):445-450. 1943. 44. Klein. M., R. Namer, E. Harpur. and R. Corbin. Earthen­ 62. Tsuchiya, K. and S. Harashima. Lead exposure and the ware containers as a source of fatal lead poisoning. New derivation of maximum allowable concentrations and England J. Med. 283( 13):669-672. 1970. threshold limit values. Brit. J. Ind. Med. 22(3):181-186, 45. Harris. R. W. and W. R. Elsen. Ceramic glaze as a source 1965. of lead poisoning. J. Am. Med. Assoc. 202(6):208-210. 63. Linch, A. L., E. G. Wiest, and M. D. Carter. Evaluation of 1967. tetraalkyl lead exposure by personal monitor surveys. J. 46. Zurlo, N. and A. M. Griffini. Lead contents in food and Amer. Ind. Hyg. Assoc. 3/(2):l70-179, 1970. beverages consumed in Milan. In: Proc. Inter. Symp. on 64. H. M. Chief Insp. of Factories. H. M. Great Britain Dept. Environmental Health Aspects of Lead, Amsterdam, Oct. of Employment. Annual Report, 1971. London, Her Ma­ 1972. Luxembourg, Commission of the European Com­ jesty's Stationery Office. 1972. p. 60, 95. munities. 1973. p. 93-98. 65. Scarlato. G.. S. Smirne. and A. E. Poloni. L'encefalopatia 47. Boudene, C. F. Arsac, and J. Meininger. Study ofair and satumina acuta dell adulto. Acta Neurol. 24:578-580. population lead levels in France. Arch. Hig. Rada. 1969. Tekisol. 26(Suppl.):179-189, 1975. 66. Maljkovic, J. A case of occupational poisoning with lead 48. Durum, W. H. Reconnaissance of Selected Minor Ele­ carbonate and stearate. Sigurnost u Pogonu. 75:123-124, ments in Surface Waters of the U.S. U.S. Dept. of Interior. 1971. Geologic Survey, Washington, D.C. USGS Circular No. 67. Sakurai. H.. M. Sugita, and K. Tsuchiya. Biological 643. 1971. response and subjective symptoms in low level lead ex­ 49. Durfor.C. N., and E. Becker. Public Water Supplies of the posure. Arch. Env. Hlth. 29:157-163, 1974. 100 Largest Cities in the United States, 1962. U.S. Dept. of Interior, Geologic Survey, Washington, D.C. USGS Water 68. Committee on Toxicology, National Research Council. Recommendations for the Prevention of Lead Poisoning Supply Paper No. 1812. 1964. 364 p. in Children. Consumer Product Safety Commission, 50. Chemical Analysis of Interstate Carrier Water Supply Washington. D.C. 1976. p. 45, 51. Systems. U.S. Environmental Protection Agency. Research Triangle Park, N.C. Pub. No. EPA 430/9-75-005. 1975. 88 69. Piver.W.T. Office of Health Hazard Assessment, NIEHS, P- Personal communication to H. L. Falk, Assoc. Dir. for 51. Rolfe, G. L. and A. Haney. An Ecosystem Analysis of En­ Health Hazard Assessment, NIEHS. Research Triangle vironmental Contamination by Lead. Inst, for Env. Park, N.C. January 10, 1977. Studies, U. of Illinois, at Urbana-Champaign, III. Res. 70. Vaughn. D. A.. C. Ifeadi, R. A. Markle, and H. H. Krause. Rept. No. 1. 1975. p. 22-34. Environmental Assessment of Future Disposal Methods 52. McCabe, L. J. et al. Survey of community water supply for Plastics in Municipal Solid Waste. U.S. Environmental systems. J. Amer. Water Works Assn. 62(11):670, 1'970. Protection Agency, National Environmental Research 53. Hem, J. D. and W. H. Durum. Solubility and occurrence Center, Cincinnati, Ohio. Pub. No. EPA-670/2-75-058. of lead in surface water. J. Amer. Water Works Assn. 1975. 86 p. 65(8):562-568, 1973. 71. Control Techniques for Lead Air Emissions. (Draft final 54. Lazrus, A. L., E. Lorange, and J. P. Lodge, Jr. Lead and report.) U.S. Environmental Protection Agency, EPA Con­ other metal ions in U.S. precipitation. Environ. Sci. Tech. tract No. 68-02-1375, Cincinnati, Ohio. October 1976. 4(l):55-58, 1970. 424 p.

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72. Hankin, L., G. H. Heichel, and R. A. Botsford. Lead on Geochim. Cosmochim. Acta. (London). 55:1247-1294, painted handles of kitchen utensils. Clin. Pediat. 1969. /5(7):635-636, 1976. 74. Jaworowski, Z. Stable lead in fossil ice and bones. Nature. 2/7:152-153. January 13, 1968. 73. Murozumi, M., T. J. Chow, and C. C. Patterson. Chemical 75. Ruhling. A. and G. Tyler. An ecological approach to the concentrations of pollutant lead aerosols, terrestrial dusts, lead problem. Bot. Notis. (Stockholme) /2/:32l-342, and sea salts in Greenland and Antarctic snow strata. 1968.

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8. EFFECTS OF LEAD ON ECOSYSTEMS

It has been substantiated that lead is a natural 8 1 EFFECTS ON DOMESTIC ANIMALS, constituent of the environment, but natural, back­ WILDLIFE, AND AQUATIC ORGANISMS ground levels of lead in the environment are not known with any degree of certainty. As a natural 8.1.1 Domestic Animals constituent, lead does not usually pose a threat to the Lead poisoning, a frequent cause of accidental organisms of natural and agroecosystems. However, death in domestic animals for many years,2 usually the widespread use of lead in a variety of chemical results from the ingestion of lead or lead-containing forms by man has redistributed the natural lead in material. Substances that cause lead poisoning in­ the environment and has consequently increased the clude lead-based paints, used motor oil, discarded exposure of the biotic components of ecosystems to oil filters, storage batteries, greases, putty, linoleum, unprecedented levels of lead. Concern now exists and old paint pails. Animals such as cattle, dogs, and about the possible threat to these biotic components cats that have natural licking and chewing habits are because oftheir inherent value to ecosystem stability particularly susceptible. Horses are not usually and because ofthe ultimate impact that effects of the poisoned in this manner because they normally do ecosystem would have on man. Figure 8-1 depicts not lick discarded materials. Animals grazing in the the environmental flow of lead and the possible ex­ vicinity of smelters, mines, and industrial plants posure routes for plants and animals in the from which lead fumes and/or dusts are being emit­ ecosystem.1 ted or that are fed vegetation harvested from such areas may also be poisoned. The possibility of animals being poisoned from ingestion of roadside pasture contaminated by mobile sources is of con­ cern, but no case of this type has been reported in the FOOD WATER 3 PROCESSING SUPPLIES literature. hr Breathing of lead dusts can be another way :•:•: PLANTS :•:•:• >':*AN'IMALS": whereby animals are exposed to lead, but poisoning of domestic and wild animals as a result of lead in­ halation has not been substantiated. LC MINING :t£ OPERATIONS Incidents of lead poisoning in cattle and horses :,,,,,,,:J—^IJ::::::::::::::::: _ "I SMELTER—S caused by emissions from stationary sources have : ATMOSPHERE > 3 2 •EARTH I (LEAD AEROSOL:; I- LAKES AND RIVERS;. : (SOIL) : AND :; been reported from Benecia, Calif.; Trail, B.C.; : ORGANIC LEAD)> Belleville, Penn.;4 St. Paul, Minn.;5 and south­ J AUTOMOBILES I »i:-4 eastern Missouri.6 Deaths from lead poisoning of w&8$. -t-f 3zm lambs and sheep in Britain7 and of horses, sheep, OTHER LEAD and goats in continental Europe have been re­ CONTAINING 8 PRODUCTS ported. -" AND WASTES 5 F4LLOUT Hammond and Aronson have estimated that the AND RAINOUT minimal cumulative lethal dose of lead for a cow is 6 to 7 mg/kg of body weight per day. They state that OCEANS -» :;ESTUARIES; this intake represents a concentration of about 300 Z3ZI ppm in the total diet. In cattle that consumed lead- contaminated hay and corn silage grown in a field Figure 8-1. Simplified ecologic flow chart for lead showing adjacent to a smelter, fatal lead poisoning occurred 12 principal cycling pathways and compartments.1 after approximately 2 months. In another study,

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cattle were fed lead at the rate of 5 to 6 mg/kg per or an increase in the amount of joint fluid in foals day for a period of 2 years without the appearance of resulted from the intake of zinc by itself and zinc and visible clinical symptoms. A steer fed the same diet lead together, but not from lead alone. Gunther,15 in for 33 months, however, showed clinical symptoms studies based on experimental exposure of colts, oflead poisoning culminating in death.13 The length reached similar conclusions. of time required for the appearance of overt clinical Horses sometimes, though infrequently, pull up symptoms of lead poisoning is in most instances plants and eat roots and soil along with leaves. This directly related to the amount consumed per unit of practice may be a factor in increasing their lead in­ time. take16 relative to that of cattle grazing the same Horses are more susceptible than cattle to poison­ pasture. ing from the chronic intake of lead. In the Trail, Lead poisoning in all domestic animals produces B.C., study,2 horses grazing near a lead smelter various degrees of derangement of the central ner­ developed overt symptoms of lead poisoning, but vous system, gastrointestinal tract, muscular system, cows in the same pasture were not clinically affected. and hematopoietic system. Younger animals appear The minimal toxic dosage for a horse has been esti­ to be more sensitive than older ones.13 Calves may mated to be between 1.7 and 2.4 mg/kg body weight suddenly begin to bellow and stagger about rolling 1 daily, which is approximately 80 ppm dry weight in their eyes, frothing at the mouth, and crashing forage. blindly into objects. This phase may last up to 2 hr, The reasons for the greater susceptibility of horses after which a sudden collapse occurs. In less severe are complex. An early symptom oflead poisoning in cases, depression, anorexia, and colic may be ob­ the horse is paralysis of the nerves of the pharynx served. The animals may become blind and may and larynx. This interferes with breathing, especially grind their teeth, move in a circle, push against ob­ on eAercise, and causes the animal to breathe ster- jects, and lose their muscle coordination. Mature torously, or to roar. When severe, this paralysis can cattle display fewer overt symptoms, although the produce suffocation and death. In addition, the syndrome of maniacal excitement is not uncom­ faulty action of the epiglottis v which closes off the mon. lung during inhalation) permits inhalation of food, Clinical symptoms in sheep consist mainly of which can result in suffocation and death or in depression, anorexia, abdominal pain, and diarrhea. severe pneumonia, which can also be fatal. Anemia is also commonly associated with lead Colts pastured near the Trail, B.C., smelter2 poisoning in sheep.1 Pavlicevic11 notes that clinical showed loss of weight, generalized muscular weak­ symptoms in lambs consist of paralysis of the ex­ ness, stiffness of joints, and harsh, dry coats. Distor­ tremities, pharynx, tongue, and larynx; a rigidly tion ofthe limbs occurred, the joints became greatly held neck; and an anemic mucous membrane. Lambs enlarged and the hocks touched. The laryngeal may be poisoned through the mother's milk when the paralysis usually associated with lead poisoning did ewe is on contaminated pasture. Sterility and abor­ not occur. Hupka9 noted the same symptoms in colts tion in ewes have been observed as a result of lead that had grazed on pasture contaminated with flue ingestion.1 dust from a metal works. Autopsy showed that the Toxic dosages of lead for domestic animals other auricular cartilages were detached and loose in the than horses and cattle have not been calculated from joint. Also noted was an acute catarrhal pneumonia statistically reliable studies. of both lobes of the lungs, with food particles in the The effects of lead on biological processes in bronchi. Roaring typical of chronic lead poisoning animals generally include effects on the nervous and did not occur until quite late. hematopoietic systems and the kidney tissue. These The clinical picture described above has come effects have been studied with various types of test under close scrutiny in various laboratory animals (primarily the rat) at the enzymatic, sub­ studies.14-15 because of the lack of consensus as to cellular, cellular, and tissue morphology levels; and whether it was the result of lead poisoning. Lead and systemically at the physiological and biochemical zinc coexist in many ores, and both were present in levels. the two situations referred to above. Under these The most sensitive indicator in rats is the decrease conditions animals being reared in the area would be of the enzyme 8-aminolevulinic acid dehydrase exposed to both elements simultaneously. Studies by (ALAD) that regulates heme synthesis.17 Lead Willoughby et al.14 indicated that lameness, blind­ clearly affects test animals at the subcellular and ness, swelling at the epiphyseal ends of long bones, enzymatic levels of biological function. P.12

8.1.2 Wildlife pellet is retained in the gizzard depends partly on its size and partly on the fiber content of the diet. A Lead has so permeated the environment that it is high fiber diet is especially conducive to lead now known to be a regularly occurring constituent of poisoning. The number of shot required to poison a all animal life. Birds and other wild animals are ex­ bird also depends on the size of the bird itself. Six posed to a wide range of lead levels. Measurable number-6 shot are always fatal for mallards, and amounts of lead may be found in the tissues of these four or five number-4 shot are generally fatal for animals, and lead poisonings have occurred. 22 Canadian geese. Toxic effects from the ingestion of spent lead shot The symptoms generally associated with lead were first observed in ducks in 1919 and have since poisoning in waterfowl are lethargy, anorexia, weak­ been recognized as a major health problem in both ness, flaccid paralysis, emaciation, anemia, greenish aquatic and upland species of waterfowl. It has been diarrhea, impaction of the proventriculus, and dis­ estimated that thousands of ducks, geese, and swans 22 18 tention ofthe gall bladder. Waterfowl appear to be die of lead poisoning each year. Lead poisoning at least twice as sensitive to the biochemical effects from spent shot has also been reported in game birds 23 19 of lead as are man and other mammals. Death ap­ such as wild pheasants, mourning doves, and quail. pears to be associated with the inhibition of The scope of the problem becomes readily apparent 8-aminolevulinic acid dehydrase (ALAD) by when it is noted that the majority of the birds die lead.23-24 In instances where insufficient lead is in­ after the hunting season is over; thus it is the breed­ gested to cause death, sterility may result.1 ing stock that is lost. Spent shotgun pellets have been In an attempt to prevent the deaths of waterfowl removed from the gizzards of birds with lead 20 through the ingestion oflead shot, the U.S. Fish and poisoning. Wildlife Service ordered the use of steel shot during Pieces of lead metal when swallowed are normally the 1976 hunting season in certain areas of the states not harmful to humans or other mammals because in the Atlantic Flyway. Despite strong opposition they pass through the digestive tract too rapidly to from the National Rifle Association and hunters, the lose more than a minor portion oftheir surfaces to plan is to extend this limitation to the Mississippi digestive enzymes and other substances. But it Flyway during 1977.25 should be noted that persons who consistently eat Waterfowl mortality caused by the toxic effects of game often have somewhat elevated blood lead lead mine wastes coupled with environmental stress levels and high fecal lead levels from swallowing was reported by Chupp and Dalke26 for the Coeur lead pellets. The gizzard of a bird, however, is a d'Alene River Valley of Idaho. Because of their comminuting organ for food that operates by grind­ feeding habits, feeding waterfowl consumed lead ing up the food in a muscular sack containing small from the sediments in the shallow areas of the river stones that the bird has swallowed. Spent shotgun along with metallic materials adhering to roots and pellets lying in the sediment on the bottoms of lakes tubers of aquatic plants. Ingestion of plants contain­ are picked up by the bottom-feeding ducks in the ing lead can also contribute to lead poisoning in same manner as pebbles. Because the shot are soft, waterfowl. they are ground fine and made quite susceptible to The puffin (Fratercula arctica), a sea bird, is in digestive action rather than just coming into super­ serious decline. In studies to determine the cause, ficial contact with the digestive tract, as in most Parslow et al.27 note the fact that puffins tend to con­ other animals.21 Lead released from the gizzard is centrate lead through the food chain (Table 8-1). absorbed by the lower digestive tract. One number-6 The authors were not able, however, to associate ob­ lead shot can furnish enough lead to induce fatal served lead concentrations with the decline of the lead poisoning in a duck; but, the length of time a species. TABLE 8-1. ACCUMULATION OF CERTAIN HEAVY METALS FROM SEAWATER BY FISH (Ammodytes AND Clupea) AND A PUFFIN"

Metal levels, ppm. wet weight Approximate accumulation tactors Fish/ Puffin/ Puffin/ Metal Seawater Fish Puffin seawater fish seawater Mercury 0.00003 0.037 0.79 1,230 21 26,300 Lead 0.00003 < 0.002 0.36 <67 >180 12,000 Cadmium 0.00011 0.309 1.67 2,800 5.4 15,000 Copper 0.003 1.74 4.49 580 2.6 1,500 Zinc 0.01 53 95 5,300 1.8 9,500

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Measurements of lead levels in pigeons28-29 and in ranged from 0.2 to 26.0 ppm. As might be expected, song birds30 indicate that urban birds have higher the highest levels were found in aquatic waterfowl; lead levels than rural birds (Table 8-2). Bagley and however, tl.." osprey (Pandion haliaetus), a predator, Locke19 analyzed 28 species of birds and noted the also showed h'°h bone levels. The bone lead levels concentration of lead in the livers and bone tissue. are an indication of continued exposure to lead and Lead in the livers was an indication of acute ex­ serve as an indication of normal levels rather than posure, and in the bone, of chronic exposure. Liver adverse exposure.19 levels ranged from 0.3 to 5.0 ppm, and bone levels

TABLE 8-2. SUMMARY OF LEAD CONCENTRATIONS IN BIRD ORGANS AND (ISSUES FROM AREAS OF HIGH-LEAD AND LOW-LEAD ENVIRONMENTS (ppm, dry weight)

Species and lead level8 Feathers Gut Liver Lung Kidney Boneb Musclec

Red-winged blackbird: Low(10) 26.5 2.1" 5.8 0.4 2.1d 6.9 0.8d High(4) 66.8 2.6d 1.2 4.1 4.1« 9.1 0.6«

House sparrow: Low(16) 27.0 2.3 0.6 0.9 3.5 16.9 0.9 High(11) 158.3 26.2 12.0 6.9 33.9 130.4 2.1

Starling: Low(11) 6.4 1.3 4.0 2.8" 3.6 12.8 0.8*1 High(13) 225.1 6.0 16.1 5.2*1 98.5 213.0 2.4"

Grackle: Low(10) 36.0 1.4 2.5 2.3" 3.5 21.5 0.8 High(11) 81.4 10.2 12.1 2.7« 13.5 62.8 1.4

Robin: Low(10) 25.3 3.2 2.4 2.2 7.3 41.3 1.0d High(10) .79.7 24.5 10.5 10.3 25.0 133.7 1.2d

a High-lead and low-lead environments are urban and rural areas respectively: high-lead area for red-winged blackbirds is 10 m from an interstate highway. Sample size in parentheses. b Femur. c Pectoral. d Differences between low- and high-lead environments not significant at the 0.05 level: all others significant at least at the 0.05 level.

Studies of lead exposure and effects in wild type of food consumed than with nearness to the animals other than birds are infrequent. Braham31 highway where the air lead concentrations were studied the distribution and concentration in the highest. California sea lion (Zalophus californianus). He One hundred and one mammals — 51 long-tailed noted that accumulation was occurring in the species field mice (Apodemus sylvalicus), 27 bank voles but could detect no adverse effects at the time ofthe (Clethrionomys glariolus), and 23 field moles study. The exposure of small mammals and selected (Microtus agrestis) — were trapped along road­ invertebrates near roadways has also been sides.32 The concentration of lead was significantly studied.32*38 In general, gradients in body lead con­ higher in Microtus than in Clethrionomys or centrations declined with increasing distance from Apodemus (Figure 8-2). The marked differences the road. The body lead gradients usually were simi­ among the lead concentrations of the three species lar in pattern to, though lower than, the soil level can be accounted for by species behavior and food gradients; but interesting exceptions were observed consumed. Microtus eats grass as its staple food, in some cases. No evidence of toxicity was observed whereas Apodemus feeds on grain, seedlings, buds, in any ofthese animals individually or in relation to fruit, hazel nuts, and animals such as snails and in­ population distributions. sects. The range of food for Clethrionomys encom­ An analysis of lead concentrations in 3 species of passes that of both the other species, but the habitat small mammals from 11 sites in Huntingdonshire, is restricted to hedges rather than the open field Great Britian,32 showed that the lead concentrations (Apodemus) or the roadside (Microtus). Differences in the animals were more closely associated with the in food and food contamination may therefore ac-

8-4 P.14

count for the differences in lead concentrations in (Microtus ochrogaster), the short-tailed shrew the three species. (Blarina brevicauda), the least shrew (Cryptotis par- va), and the white harvest mouse (Reithrodontomys megalotis) — had higher total body lead burdens and higher levels in selected tissues than did those species such as the white-footed mouse (Peromyscus FIELD MOLES (Microtus) maniculatus) and the house mouse (Mus musculus) BANK VOLES that extend their home ranges into cultivated fields. (Clethrionomys) FIELD MICE There was also a correlation between feeding habits (Apodemus) and lead concentrations in body tissues. Insectivores D-MEAN OF SPECIES (the shrews) had the highest lead concentrations; herbivores (voles) had intermediate concentrations; and granivores (deer mice, white-footed mice, and house mice) had the lowest concentrations, reflect­ ing the fact that lead concentrations in seed tissue are usually extremely low (< 1 ppm). It is highly doubtful that the very low concentra­ tions of lead in these mammals (Table 8-3) could be having a significant impact on their population dynamics. Studies of lead concentrations in insects in central Illinois ecosystems have shown positive correlations with lead emission levels, decreasing from areas ad­ jacent to heavily traveled roads to areas remote MEAN Pb ON VEGETATION from roads (Table 8-4).39 There was also a strong 306.7 ppm dry wt. 42.5 ppm dry wt. 33.4 ppm dry wt. trend of increasing lead content from sucking to chewing to predatory insects collected near high- TYPE OF SITE traffic roadways (Figure 8-3). Chewing insects pro­ Figure 8-2. Concentration of lead In three species of small bably ingested more lead from deposits on leaves mammals trapped beside major and minor roads and at ara­ than did insects that suck liquids from the internal ble and woodland sites.33 vascular tissues of plants. Data on predatory insects In a study in central Illinois, samples of small that feed on lead-containing herbivores suggest that mammals were obtained from a range of environ­ lead is selectively retained in the body, leading to ments including those within 10 m ofa high-traffic- biological concentration in this two-trophic (feed­ 39 volume road (> 12,000 vehicles/24hr), those within ing-level) system. 5 m of medium-use roads (2000 to 6000 vehicles/24 Definitive studies correlating toxicity with en­ hr), those within 5 m of low-use roads (<2000 vehi­ vironmental lead concentration have not been done. cles/24 hr), and those in urban areas (approximately 8.1.3 Aquatic Organisms 100,000 inhabitants).38 Acute lead toxicity in aquatic organisms has been All species except the white-footed mouse observed and studied experimentally. Lead toxicity (Peromyscus leucopus), showed higher concentra­ in fish is partially related to drainage from metallic tions of lead in habitats adjacent to high-traffic- wastes into streams. Early experiments were carried volume situations, especially in urban areas. Since out in England where contamination of natural the home range of this species averages more than 50 waters by lead mining caused the disappearance of m in diameter, even those individuals caught nearest fish from streams.1 Although the effect of lead on the highway were undoubtedly spending considera­ lower forms of life is not well documented, it ap­ ble time much further removed from the traffic pears to be less toxic than in higher forms.' •40 source, possibly accounting for the low lead levels in these animals. Apparently, lead and other metals are irritating to There was also a correlation between habitat re­ the skin of many freshwater fish and cause an quirements and lead concentrations in small mam­ unusual reaction. The presence of metal in the water mals captured near high-traffic roadways. Species around them causes a copious secretion of mucus requiring dense vegetation—the prairie vole over the whole body surface, particularly in the gill

8-5 P.15

TABLE 8-3. MEAN LEAD CONCENTRATIONS IN ORGANS AND TISSUES OF SMALL MAMMALS FROM INDICATED AREAS OF ENVIRONMENTAL LEAD EXPOSURE3' (ppm, dry weight)

Species and Total exposure area body Gut Spleen Liver Lung Kidney Bone8 Muscle6

Blarina brevicauda: High 18.4 24.0 4.5 4.6 16.9 12.4 67.1 9.7 Medium 6.7 7.0 3.6 2.0 5.6 5.8 19.9 5.7 Low 5.7 3.1 2.3 1.0 7.8 3.9 12.2 5.4

Microtus ochrogaster: High 5.1 11.0 5.3 1.6 2.8 8.1 16.6 8.2 Medium 5.9 18.4 2.2 1.2 1.8 7.6 23.2 3.0 Low 1.9 2.8 2.4 1.0 1.3 2.8 4.6 2.0 Peromyscus maniculatus: High 6.3 19.2 19.4 3.5 6.4 7.9 24.6 6.8 Medium 4.3 6.0 3.0 1.7 2.4 9.0 8.0 7.4 Low 3.3 4.5 6.5 1.8 6.1 3.0 6.4 1.8 Control0 3.1 4.3 3.7 1.1 1.5 1.8 5.7 2.1 Mus musculus: High 6.8 18.6 12.1 2.9 2.8 8.1 19.2 5.9 Medium 6.0 8.8 3.1 1.6 3.4 6.6 21.0 3.9 Low 6.7 4.8 5.1 1.6 1.7 3.1 23.5 3.4 Control0 2.0 2.7 2.1 1.9 3.4 3.4 9.3 3.8 Reithrodontomys megalotis: High 12.3 17.8 145 4.7 20.9 — 109.5 27.5 Medium 3.0 6.2 9.2 1.1 4.2 2.1 — 4.4 Low 2.7± 3.5 5.6 2.3 4.7 4.8 18.4 — a Femur. "Thigh. c Control areas are fields more than 50 m from a road. TABLE 8-4. LEAD CONCENTRATIONS OF INSECTS AT TWO Symptoms of chronic lead poisoning in fish in­ DISTANCES FROM A HIGH-TRAFFIC-VOLUME ROAD clude anemia, functional damage to the inner (INTERSTATE HIGHWAY)3" organs, possible damage to the respiratory system, (ppm) growth inhibition, and retardation of sexual maturity.1-42 0 to 7 m from 13 to 20 m from 43 Feeding type pavement pavement A study in central Illinois of both urban and rural tributaries of the Saline Branch of the Ver­ Sucking 15.7 9.8 milion River showed that lead appears to be taken Chewing 27.3 10.4 Predatory 31.0 20.0 up by aquatic organisms by means of external con­ Mean 24.7 13.4 tact rather than by ingestion. Lead concentrations in aquatic organisms were found to be related to the area. Analysis of this mucus has revealed the pre­ 41 amount of contact with substrates, such as sediments sence of "considerable quantitities" of lead. The (Figure 8-4), that contain the highest lead con­ mucus does not, however, prevent absorption of lead centrations in the streams. Thus species differences into the fish. If the metal level is low, the process is in Concentrations are determined in part by habitat harmless because the excreted film is readily shed; preference and feeding habits. but if higher levels are present, the mucus blanket generated may suffocate the animal before it can be Filtered water from the two streams had con­ shed. The phenomenon has been observed only in centrations of lead varying from 0 to 15 mg/liter of freshwater fish and is subject to modification by water (ppm), and suspended solids in the water con­ water hardness, temperature, and other factors. It tained 15 to 200 ppm lead. The highest levels occur­ should be emphasized that the process described red in the urban stream. The upper 10 cm of sedi­ above is entirely external and unrelated to lead ments in the urban stream contained an average lead levels in the body of the fish. In some cases of lead concentration of 387.5 ppm, more than 10 times poisoning in freshwater fish, mucus formation has greater than that in the rural stream. Fish in the not been observed. rural stream contained an average of 1.4 to 4.1 ppm

8-6 P.16

I STD. ERROR """"") URBAN COMPARTMENT D LOW Pb EMISSIONS AREA *"*""* COMBINED COMPARTMENT CD HIGH Pb EMISSIONS AREA I I MARGINAL COMPARTMENT "• RURAL MAIN CHANNEL I"""*" RURAL TRIBUTARIES

SEDIMENTS CLADOPHORACEAE ii TUBIFICIDAE CHIRONOMIDAE

CHEWING PREDATORY SEDIMENTS ANO ORGANISMS

INSECT FEEDING TYPES Figure 8-4. Mean lead levels in representative organisms and Figure 8-3. Mean lead content In grouped Insect samples sediments of the compartments of the drainage basin of the taken from low- and hlgh-lead-emlsslon areas for sucking, Saline Branch of the Vermilion River, III.43 chewing, and predatory feeding types.39

being 482 and 442 mg/liter, respectively;47 whereas of lead in dry tissue (Table 8-5).43 No fish were for rainbow trout, Davies and Everhard42 report found in the urban stream. Lead levels in the inver­ 471 mg/liter. Detrimental effects on fish species oc­ tebrates ranged from about 5 to 20 ppm in the rural cur at concentrations as low as 0.1 mg/liter. In stream to more than 350 ppm in the urban stream. studies of rainbow trout, mortalities attributed to These data appear to be in agreement with relative lead occurred at the high test concentrations, which lead levels found in tubificid worms, clams, and fish in soft water were 95.2 /xg Pb/liter and in hard in the Illinois River.40 water, 3.24 mg/liter total lead or 0.064 mg/liter free Hardisty et al.44 studied lead levels in estuarine lead.42 Physical abnormalities occurred between fish, but were unable to determine any biological 11.9 and 6.0 fig Pb/liter in soft water and between effects of lead. Merlini and Pozzi45 studied lead ac­ 0.12 and 0.36 mg/liter total lead (0.018 and 0.032 cumulation by freshwater fish. Ionic lead (as Pb + +) mg/liter free lead) in hard water. In Daphnia magna, was concentrated threefold when the pH ofthe lake an effect on reproduction has been observed at 0.03 water was lowered. Lead toxicity in fish was not re­ mg Pb/liter.48 ported. A study of long-term effects of lead exposure on Toxicity varies with pH, temperature, hardness, three generations of brook trout (Salvelinus fon­ and other water properties.40 The concentrations of tinalis) indicated that all second generation trout ex­ lead injurious to fich and other aquatic and marine posed to 235 and 474 fig Pb/liter developed severe life may be found in Water Quality Criteria, 1972.4(l spinal deformities (scoliosis), and 34 percent of The 96-hr LC50 value in soft water for rainbow trout those exposed to 119 fig Pb/liter developed (Salmo gairdneri) has been reported to be 1 mg/liter. scoliosis. Of the newly hatched third generation 47 Pickering and Henderson list the soft water LC50 alevins exposed to 119 fig Pb/liter, 21 percent values for fathead minnows (Pimephales promelas) developed scoliosis. Analysis of residues in eggs, and bluegills (Lepomis macrochlrus) as being 5 to 7, alevins, and juveniles indicated that accumulation of and 23.8 mg/liter, respectively. In hard water, the lead occurred during these lite stages.49 50 LC50 values for the last two species are reported as Weir and Hine utilized a conditioned avoidance P.17

TABLE 8-5. LEAD CONCENTRATIONS OF PREDOMINANT TABLE 8-6. SUMMARY OF GOLDFISH TOXICITY DATA")

ORGANISMS FROM THE RURAL, URBAN, AND COMBINED ab h Ion LC50. ppm LCi.ppm Slope COMPARTMENTS OF THE DRAINAGE BASIN OF THE SALINE BRANCH OF THE VERMILION RIVER, ILL." Arsenic 32.0 (41.6 to 24.6) 1.5 1.4 (ppm, dry weight) Lead 110.0(121.0 to 100.0) 60.0 1.2 Lead (without Arithmetic Number of Standard Compartment and organism mean samples deviation calcium carbonate) 6.6 (9.2 to 4.7) 1.5 1.6 Mercury 0.82 (0.90 to 0.75) 0.36 1.2 Rural: Selenium 12.0 (17.0 to 7.9) 1.0 2.5 Plants: a Based on a 7-day survival time. Cladophora 20.1 11 5.1 ^Lowest concentrations of ions that significantly impaired conditioned avoidance Potarrrgeton 30.0 15 5.4 responses and their fractions of the 50- and 1-percent lethal concentrations. . InvertebrtiTf s: Two species of marine gastropods were shown to Hirudinea 12.6 12 14.8 concentrate lead in their shells.51 However, Valiela Oligochaeta 13.2 7 7.6 et al.5'2 reported that three species of estuarine Tubificidae 16.0 13 11.8 Sphaeriidae 5.5 11 3.1 bivalves showed no increase in lead when sludge fer­ Lirceus fontinalis 7.6 5 3.4 tilizer containing lead was added to experimental Hexagenia limbata 10.4 15 9.7 plots in a salt marsh. Furthermore, the lead ap­ Anisoptera 6.6 6 2.4 parently was trapped by the marsh surface sedi­ Chironomidae 20.1 12 18.2 ments, since no increases were recorded downstream Decapoda 4.7 23 5.2 in the detritus on creek bottoms or in filter-feeding Fish: bivalves. Lead uptake by the cordgrass Spartina Catostomus commersoni (white sucker) 2.4 19 1.6 alterniflora reduces by a small amount the quantity 53 Etheostoma nigrum of lead in the sediments. (Johnny darter) 4.1 9 2.4 Aquatic lead concentrations of 0.5 mg/liter have Ericymba buccata been shown to be toxic to flagellates and infusoria, 0.9 (Silverjaw minnow) 1.8 29 the microscopic animals that frequently develop in Notropis umbratilus (Redfin shiner) 1.9 29 0.6 decaying organic matter. Bacterial decomposition of Pimephales notatus organic matter is inhibited by lead concentrations of (Bluntnose minnow) 2.7 83 2.7 0.1 to 0.5 mg/liter.54 Semotilus atromaculatus (Creek chub) 1.4 35 0.6 8.2 EFFECTS ON PLANTS Urban: 8.2.1 Routes of Plant Exposures Plants: Claoophoi . 347 139 Plants may be exposed to lead through the leaves, Invertebrates. stems, bark, or roots. The extent of exposure de­ Tub' cidae 367 29 373 pends on the amount of lead in the immediate en­ Chironomidae 153 5 106 vironment and the form and availability of that lead. Decapoda 11 4 8.2.1.1 LEAVES, STEMS, AND BARK Combined: Plants: Particulate substances in the air, including lead, Elodea 89.9 22 93.3 are deposited on plant surfaces by fallout, impac­ Cladophora 34.9 7 7.5 tion, and precipitation.55 Precipitation may, in addi­ Invertebrates: tion to depositing material, wash off or leach out Tubificidae 48.6 71 33.9 material from the plant surface or tissue. Wind and Chironomidae 42.7 12 16.4 Physa 41.7 10 25.5 sloughing of the cuticle wax may also remove Psv,chodidae 32.3 4 25.5 deposited material. Thus, meteorological factors are important in determining the fate of compounds that come into contact with plant surfaces. The technique to assess the deleterious effects of four morphology ofthe plant surface, however, plays the metal ions, including lead, on goldfish. Behavioral major role in determining the type and quantity of impairment was noted following sublethal con­ mateiial that will be retained by that plant sur­ centrations of lead nitrate (Table 8-6). The lowest face.5657 Epidermal cellsof the aerial parts of plants concentration that gave significant impairment was are usually coated with a waxy substance, cutin, and 0.07 ppm. often form unicellular or multicellular hairs, spines, Shellfish have been reported to concentrate lead.1 or glands. In addition, secretions from glandular hairs may make leaf surfaces sticky; thus, particulate burden. The complexities of lead movement from material can accumulate on the plant surface, partic­ soil into plants make it unlikely that plants are capa­ ularly the leaves. However, particulate matter must ble of exuding large amounts into the air. 56 enter the internal plant tissues to affect the plant. Particles containing lead, in addition to chlorine 58 Franke suggests that the cutin is penetrable via in­ and bromine, have been found embedded in the bark termolecular spaces and that cuticle has been shown of both pine and elm trees growing near highways. to be permeable to both organic and inorganic ions The lead content of the particles on the trees and to undissociated molecules. The capability of an decreased over a 3-year period, and the particles on ion to penetrate is determined by its charge, adsorb- the bark of elm trees showed less lead than those on ability, and radius. pine trees.63 No evidence exists to show that lead en­ Vascular plants have small surface openings, tered the trees through the bark. called stomata and lenticels, that function in gas ex­ Trees have been used as indicators of increasing change. Stomata are also sites of exchange of environmental lead concentrations with time.64 58 aqueous substances under certain conditions. Studies in central Illinois have shown a fivefold in­ Stomata occur in the epidermis of leaves and young crease in lead in tree rings during the last 50 years stems. Lenticels are found on older stems of woody (Figure 8-5). This graphically illustrates the increase species. Surrounding the stomatal pores are guard in environmental lead uptake that has occurred in cells that open and close the pores through changes that time. However, data reported by Holtzman on in their turgidity. Gas exchange, which takes place four hardwood trees in a rural suburban area and on through the stomata as the result of a concentration one tree in a suburban area near Chicago (tree ages gradient, is a passive phenomenon unlike the active were 100 to 120 years) showed no consistent in­ breathing of animals. crease or decrease in lead in the tree rings.65 Large deposits of inert, insoluble metal com­ pounds on the leaves are probably of little conse­ quence to a plant. The most important factor in 10-year AVERAGE, determining foliar penetration is the solubility ofthe 1963- 1973 individual metal.5658 The insolubility oflead is un­ doubtedly a major reason that little incorporation and accumulation occur through the leaf surface.56 Carlson et al.57 experimentally fumigated soybean (Glycine max L.) with PbCl 2 aerosol particulate; no intraplant movement was noted. Simulated rainfall removed up to 95 percent of the topically applied lead. Studies by Arvik and Zimdahl59-60 indicated that only extremely small amounts of lead could penetrate plant cuticles, even after extended ex­ posure. Increased penetration of some cuticles oc­ curred after the removal of waxes, but penetration seemed more related to plant species than to cuticle thickness. Oats (Avena sativa L.) and lettuce {Lactuca sativa var. Black-Seeded Simpson) are two species, according to Rabinowitz, whose leaves atmospheric lead is capable of penetrating.61 The only conclusion possible from current data is that airborne lead can be taken up by the foliage of some plants, but only in extremely small amounts. 62 Figure 8-5. Tree ring analysis of lead concentrations in urban A recent report suggests that grasses and small and rural trees.63 herbaceous plants in the field, as well as pea plants (Pisum sativum L.) and pine tree seedlings (Pinus 8.2.1.2 ROOTS sylvestris L.) in the laboratory, when grown in The root system is the major pathway of plant ex­ nutrient solution, release lead and zinc into the air. posure to lead, as lead in the soil may be absorbed by It is questionable whether the lead exuded from the roots and moved into the plant.6066"77 But the plant leaves adds appreciably to the atmospheric total lead content ofthe soil is only one of several in-

8-9 P.19

teracting factors determining plant uptake. These tle is known, however, about the mechanism of lead factors are not well understood, but uptake is known uptake. Undoubtedly, lead in solution moves into to be influenced by plant species and by the availa­ the plant through the root hairs along with mineral ble lead pool in soils.66-7174 The pool of available nutrients and is translocated to other areas of the lead is determined by soil pH, soil organic and clay plant. The vascular tissue is the pathway of water in fractions, and the cation exchange capacity as well as the root, through the stem, into the petioles, and into other soil sorption characteristics.66-70*8' Absorption the leaf veins.89 After entering the root hairs, water of lead by plant roots is inversely related to cation containing lead and nutrients must pass through the exchange capacity.74-79-80 The total lead in the soil in root cortex to reach the central core of vascular relation to cation exchange capacity determines lead tissue. Because the movement of water through the availability.80 Roots take up minerals that are in the cortex is from cell to cell with no specific pathway soil solution;60-82 thus lead movement into roots is in such as vascular tissue, lead possibly may not pass the form of ions from the soil solution or from weak easily through cell membranes. This may explain sorption sites.60-66-74 why plant roots show higher lead contents than other 90 Baumhardt and Welch70 have shown that the per­ plant organs. Malone et al. have shown that some centage of soluble lead in soil decreases with time as ofthe lead that enters the root is concentrated in dic- sorption occurs. Soil phosphate60-75-77-78 and lim- tyosome vesicles and subsequently moves via the ing,60-75-83-84 as well as cadmium85 in soil, reduce the vesicles to the cell wall, where fusion with the cell uptake of lead by plant roots. Olson and Skoger­ wall occurs. boe86 have identified the primary lead species in Lead has been reported to have both a beneficial their soil tests as lead sulfate (Chapter 6). At pres­ and an inhibitory effect on plant growth.66-69 ent, the capability of plants to take up this relatively Brewer69 cites studies in which lead nitrate resulted insoluble form is not understood.74 Jones et al.87 in increased nitrification and increased plant growth have reported that lead uptake is enhanced by a defi­ when added to the soil. However, when lead nitrate ciency of sulfate in the soil. In this study, the lead in was added to solution cultures, retardation of root rye grass tops was higher than that in the roots when growth occurred. In neither case was the metabolic the soil was deficient in sulfur. action of lead nitrate observed in the plants. The Once lead enters the plant from the soil solution, chemical identity of lead in plants is as yet not most of it remains in the roots.60-66-72-77 Distribution known.60 to other portions of the plant does occur, but it is Most studies60-66-69 that describe growth inhibition uneven and variable among species.71-72-77 Plant age and plant toxicity caused by lead compounds are and season of the year also affect internal distribu­ based on visible growth responses resulting from tion.66-72 In all cases, the levels are quite low because lead added to soils or to solution cultures and do not of the small amounts of available lead in the soil deal with specific metabolic processes. solution. The effects of lead compounds on such plant pro­ cesses as photosynthesis,60-91-93 mitosis,'-60-66 and 8.2.2 Effects on Vascular Plants water uptake92 have been reported. Miles et al.,91 Lead is a normal soil constituent, but it has not using isolated chloroplasts from spinach leaves, been shown to be essential for plant growth.66-69 The found that lead salts inhibit photosynthetic electron response of plants to lead is therefore dependent on transport. Wong and Govindjee94 have shown in the extent to which normal metabolic processes are isolated maize chloroplasts that lead salts affect disturbed. Metabolic disturbances manifest them­ photosystem I, inhibiting P700 photooxidation and selves as growth abnormalities (the visible symptoms altering the kinetics of re-reduction of P700. In of which may be growth stimulation, stunting, laboratory studies, lead has been found to have a yellowing or purpling of leaves) or, in the event of damaging effect on cell walls, nuclei, and acute toxicity, senescence and death.82 Metabolic mitochondria. Lead retarded cell proliferation but disturbances are most likely to occur in response to permitted an increase in size.1 Spindle disturbances high available lead levels and to highly soluble and chromatid formation in root tips of Allium cepa forms of lead entering the plant.66-69 induced by lead nitrate have been found to be in­ Antonovics, Bradshaw, and Turner88 state that distinguishable from those induced by colchicine.' lead uptake is constant with increasing levels of soil Miller and Koeppe95 and Bittell et al.96 studied lead until a certain point is reached at which lead the effects of lead on mitochondrial respiration. uptake becomes unrestricted and rises abruptly. Lit­ Lead effects are related to the phosphate status of

8-10 the respiratory system as well as to the oxidation of system for uptake of nutrients from soil or other nicotinamide adenine diphosphonucleotide substrates. Mosses also have neither epidermal cells monohydrogen (NADH). The lead levels used in nor a cuticle (waxy layer), so the internal this study approximated the levels found near parenchymal cells are readily exposed to substances heavily traveled highways. The form of lead used, from the air.99100 Accumulation of heavy metal ions PbC 12, and the isolated mitochondria were not typi­ in mosses is generally from precipitation, which is a cal of field conditions, but lead in plants does associ­ very dilute solution of metals and water. The ac­ ate with mitochondria and chloroplast cumulation occurs because ofthe chemical complex­ 60 66 75 membranes. - - es formed between these heavy metal ions in pre­ The presence of lead in plants has also been shown cipitation and negatively charged organic growth.101 to have indirect effects on plant growth. For exam­ Lichens, like mosses, have no roots; therefore all ple, absorption of phosphorus and manganese, two minerals are absorbed through the cell membranes. essential elements for growth, is inhibited when lead Mechanisms similar to those found in mosses may is present in the plant.97 Lead may also contribute to also be responsible for the uptake of metal ions by copper deficiency in plants.98 lichens. But lichen accumulation of lead is not as ex­ The majority ofthe studies reporting lead toxicity tensive as that in mosses.I01 have been conducted with plants grown in artificial 8.2.3.2 ALGAE nutrient culture. As a result of the studies, the con­ cept has emerged that the effects of lead, whether Trollope and Evans,102 in a study of algal blooms stimulatory or inhibitory, depend on a variety of en­ in the Lower Swansea Valley, Wales, noted the sen­ vironmental factors, including associated anions and sitivity of algae to the heavy metal content of water. cations within the plant and in the growth media, A marked difference was observed in the nature of and the physical and chemical characteristics ofthe algal blooms found in three different groups of soil itself. Because lead interacts with so many en­ waters at 12 different stations (Table 8-7). The algae vironmental factors, specific correlations between most tolerant to high lead concentrations were: Coc- lead effects and lead concentrations are extremely comyxa, Mougeotia, Tribonema, and Zygnema. Less difficult to predict.60 tolerant were Microspora, Oscillatoria and Ulothrix, Lead toxicity has not been observed in plants and the least tolerant were Cladophora, Oedogonium, growing under field conditions. This observation and Spirogyra. All ofthese algae were subject to con­ may be explained by the fact that ambient lead con­ tamination from run-off water and dust from the centrations in the environment have not been high zinc smelter. The concentrations of lead in the plants enough, except under unusual conditions (near varied among genera and within a genus. The uptake mines and smelters) to cause a toxic effect or a of individual metals by algal blooms appears to be decrease in crop yield.60 regulated. Mean metal concentrations in the three In summary, the effects of lead on vascular plants groups of algae are ordered Fe >Zn >Pb >Cu appear at this time to be minimal. The most impor­ >Ni, whereas the mean metal concentrations in the tant effects may be those resulting from ingestion of aquatic bodies were ordered differently: Adjacent to topical and internal plant lead by grazing animals the source, Zn >Pb >Fe >Ni >Cu; near, Zn >Ni (the next trophic level). From the standpoint of eco­ >Pb >Fe >Cu; distant (6 to 10km),Fe >Zn >Ni, nomic consequences, evidence developed on the Pb >Cu. Concentrations of metals in the algae were effects of lead in agroecosystems indicates that topi­ directly related to the concentrations in the water, cal lead contamination of plants is more likely to with the algae in the most polluted waters having the have economic consequences than internal lead (see highest concentrations. No experiments were con­ Section 6.4.3). ducted to determine whether the algae found in the least polluted waters would grow in more polluted 8.2.3 Effects on Nonvascular Plants ponds.

8.2.3.1 MOSSES AND LICHENS Observations in the New Lead Belt of south­ Mosses have been shown to have unique capacity eastern Missouri103 have shown that relatively high for sorbing heavy metal ions to their surfaces. Traces concentrations of lead in stream bottom sediments of copper and lead are sorbed readily even in the do not have much effect on algal growth in these re­ presence of other metal ions (calcium, magnesium, latively hard natural waters. Under these conditions, potassium, sodium).99 These ions are sorbed through the dissolved lead salts are in very low concentra­ the leaves of the moss because mosses have no root tions and well below the limits of tolerance of most

8-11 P.21

TABLE 8-7. CONCENTRATIONS OF SELECTED HEAVY METALS IN FRESH WATER AND IN FRESHWATER ALGAL BLOOMS""

Concentrations in fresh waters. \i g 'ml Concentrations in freshwater algal blooms, M 9'mg Area of water Fe Ni Pb Algal bloom Cu Fe Ni Pb Zn

Water adjacent to zinc smelting waste: 1 0-03 0-24 0-15 0-31 34-1 Mougeotia 0-38 17-61 0-24 6-19 44-94 Tribonemaa 0-4 9-97 0-16 5-45 19-93 Tribonema b 0-7 33-92 0-26 4-94 17-61 2 0-02 0-11 0-1 1-24 19-61 Tribonemac 0-67 23-37 0-29 3-68 21-11 Tribonema d 1-33 30-6 0-24 14-19 17-44 3 0-01 0-25 0-12 0-1 11-44 Coccomyxa 0-65 49-51 0-15 3-23 19-05 Zygnema 0-46 39-85 0-7 2-6 45-89 4 0-02 0-28 0-15 0-1 1-96 Mean 0-02 0-22 0-13 0-44 16-78 Mean 0-66 29-26 0-29 5-75 26-57 Water near zinc smelting waste: 5 0-02 0-27 0-12 0-1 1-96 Oscillatoria 0-34 2-8 1-07 0-58 1-88 6 0-05 0-56 2-2 0-31 4-9 Ulothrix 0-48 7-78 0-3 2-38 3-56 7 0-06 0-56 2-94 2-91 4-9 Microspora 1-02 42-31 0-11 2-16 9-26 Mean 0-04 0-46 1-75 1-11 3-92 Mean 0-61 17-63 0-49 1-70 4-89

Water distant from zincsme Iting waste: 8 0-03 0-39 0-07 0-1 0-21 Cladophora a 0-06 3-94 0-03 0-23 0-89 Spirogyra a 0-22 3-03 0-13 0-4 1-59 Spirogyrab 0-29 7-66 0-12 0-11 1-92 9 0-02 0-1 0-06 0-1 0-08 Oedogonium 0-11 0-7 0-07 0-06 0-12 10 0-01 0-39 0-12 0-1 0-08 Cladophora b 0-05 2-91 0-1 0-09 0-97 11 0-02 0-39 0-12 0-1 0-16 Spirogyra c 0-23 0-46 0-09 0-13 1-09 12 0-02 0-11 0-12 0-1 0-05 Spirogyra d 0-05 8-93 0-03 0-04 0-32 Mean 0-02 0-28 0-1 0-1 0-12 Mean 0-14 3-95 0-08 0-15 0-98

algae, including the sensitive Cladophora. Extensive lead (Me4Pb) when the sediment was enriched with blooms of Cladophora were observed in one stream nutrient broth and glucose to stimulate growth and where bound lead associated with the filaments ex­ growth occurred under anaerobic conditions. The ceeded 5000 ppm. These results indicate that lead Me4Pb lead production was greatly increased when chelated or bound to the cell envelopes apparently inorganic lead nitrate or organic trimethyl lead ace­ had no major physiological effect on algae under the tate (Me3PbOAc) was added at 5 mg per liter of existing natural conditions. sample. The biological methylation from trimethyl lead (Me Pb) to Me Pb appeared to proceed quite 8.2.3.3 BACTERIA 3 4 readily. This conversion was demonstrated using The response of certain bacteria to lead has been pure species of bacterial isolates from lake sediments studied by Tornabene and Edwards.104105 Micrococ­ without the sediments being present. They were able cus luteus and Azotobacter sp., when grown in lead- to show that Pseudomonas, Alcaligenes, containing media under experimental conditions, Acinetobacter, Flavobacterium, and Aeromonas sp. were able to take up substantial quantities of lead growing in a chemically defined medium could with no apparent effects on cell viability. Most ofthe transform lead nitrate, lead chloride, and trimethyl lead became associated with the cell membrane. Sev­ lead acetate (Me3PbAc) into volatile tetramethyl 106107 eral studies indicate that bacteria in lake sedi­ lead (Me4Pb). None ofthe bacteria were able to con­ ments under anaerobic conditions react differently. vert insoluble lead to Me4Pb. Schmidt and Methylation of mercury and arsenic by microorgan­ Huber,107 however, have observed that Pb2+ can be 108109 isms is a well-known phenomenon, but the biologically alkylated and transformed to Me4Pb methylation of lead is not. The first evidence for the (see Section 6.4.2.2). methylation of lead was demonstrated experimen­ Generally, most naturally occurring bacteria can 110 tally by Wong et al. Microorganisms in lake sedi­ tolerate lead without toxicity.'04"0 However, there ments were able to transform certain inorganic and is wide variation in effects. For example, lead has organic lead compounds into a volatile tetramethyl been shown to stimulate growth ofa bacterium iden-

8-12 tified as Micrococcusflava Strevisan,"' producing an Lead, cadmium, zinc, and copper were the primary insoluble lead metabolite; but lead has also been elements studied. Litter mass, heavy metal and shown to be widely inhibitory to aerobic activated macronutrient content (Ca, P, K, and Mg), cation sludge bacteria,"2 aerobic river water bacteria,"3 exchange capacity, and pH were studied to charac­ and marine sulfate-reducing bacteria."4 These re­ terize the arthropod food base. Arthropods were ports seem to indicate that lead has a relatively removed from litter by Von Tullgren funnel extrac­ casual relationship with bacterial cells, with no tion. The arthropods were taxonomically classified specific inhibitory role; but this should be viewed according to their feeding habits or levels: with caution. detritivore, fungivore, littergrazer, omnivore, and Previously, the presence of lead in estuarine sedi­ predator. Level refers to the sequential location ofa ments was mentioned. The methylation of lead in particular organism in the food chain or web. Their this environment and its effects on the biota existing population density at each trophic level, biomass, there have not been studied. and heavy metal and macronutrient content was Effects of the addition of 1000 ppm of copper, determined. nickel, lead, and zinc on carbon dioxide release dur­ Changes in litter decomposition and nutrient cy­ ing aerobic incubation of soil alone and after treat­ cling were reflected in the population dynamics of ment with straw were studied.115 Carbon dioxide re­ litter arthropods and macronutrient pools. Reduced lease during incubation from soils without added arthropod density, biomass, and richness (an esti­ straw was decreased by all metallic elements. Car­ mate of maximum diversity) were observed. The bon djoxide release from soil plus straw was macronutrients Ca, K, and Mg in the 01 and 02 litter decreased by lead. The toxic effects ofthe high con­ layer at a site 0.4 km from the smelter were signifi­ centration of elements on the activity of the cantly reduced. Two litter layers or horizons are microorganisms attacking organic matter were recognized by the Soil Science Society of America. believed to be caused by the ability of the elements The 01, or surface layer, is that in which dead plant to compete with essential elements (manganese, iron, material still retains its original conformation. The and zinc) for the active sites (SH, NH2, = NH) of 02 layer is that layer in which the material is frag­ enzymes. Nickel and lead were slightly more in­ mented and no longer recognizable as to species or hibitory than copper and zinc.115 origin. Cole'16 found that addition of lead to soil resulted Mean heavy-metal concentrations were greater in in 75- and 50-percent decreases in net synthesis of the undecomposed 02 litter layer collected at 0.45 amylase and a-glucosidase, respectively. The and 0.8 km from the smelter. The Pb concentration decrease in amylase synthesis was accompanied by a was 103,000 ppm; Zn was 4910 ppm; Cu was 6080 decrease in the number of lead-sensitive, amylase- ppm; and Cd was 179 ppm. At these sites, heavy- producing bacteria, whereas recovery of synthesis metal concentrations correlated with 02 litter layer (usually in 24 to 48 hr) was associated with an in­ accumulations. A change from the normal was also crease in the number of amylase-producing bacteria, noted in the cation exchange capacity and pH of the presumably lead-resistant forms. The results indi­ soil. cated that lead is a potent but somewhat selective in­ In summary, the results of this study117 indicate hibitor of enzyme synthesis in soil and that highly in­ that the dynamics of forest-nutrient cycling pro­ soluble lead compounds such as PbS may be potent cesses are seriously disturbed near these lead modifiers of soil biological activity. smeltering complexes.

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Jarvis, and D. W. Cowling. Lead up­ viron. Sci. Tech. 4(3):217-223, 1970. take from soils by perennial ryegrass and its relation to the 68. Ter Harr, G. Air as a source of lead in edible crops. En­ supply of an essential element (sulphur). PI. Soil. viron. Sci. Tech. 4(3):226-229, 1970. 55:605-619, 1973.

8-15 88. Antonovics, J., A. D. Bradshaw, and R. G. Turner. Heavy 103. Gale, N. L., P. Marcellus, and G. Underwood. The impact metal tolerance in plants. Adv. Ecol. Res. (London) oflead mining and milling activities on aquatic organisms. 7:1-85, 1971. In: Trace Contaminants in the Environment, Proc. 2nd 89. Lepp, N. W. The potential of tree-ring analysis for Ann. NSF-RANN Trace Contaminants Conference. monitoring heavy metal pollution patterns. Environ. Poll. Asilomar, Pacific Grove, Calif. 1974. p. 295-307. 9:49-61, 1975. 104. Tornabene, T. G. and H. W. Edwards. Microbial uptake 90. Malone, C, D. E. Koeppe, and R. J. Miller. Localization oflead. Sci. / 76:1334-1335, 1972. of lead accumulated by corn plants. Plant Phys. 105. Tornabene, T. G. and H. W. Edwards. Effects of lead on 54:388-394, 1974. bacterial membranes. In: Trace Substances in Environ­ 91. Miles, C. D., J. R. Brandle, D. J. Daniel, O. Chu-Der, P. mental Health. Vol. VII. Proc. of U. Missouri's 7th Ann. D. Schnare, and D. J. Uhlik. Inhibition of photosystem II Conf. on Trace Substances in Environmental Health. in isolated chloroplasts by lead. Plant Physiol. University of Missouri, Columbia, Mo. 1974. p. 263-266. 49:820-825, 1972. 106. Jarvie, A. W. P., R. N. Markall, and H. R. Potter. Chemi­ 92. Bazzaz, F. A., G. L. Rolfe, and P. Windle. Differing sen­ cal alkylation oflead. Nature. 255(5505):2I7-218, 1975. sitivity of corn and soybean photosynthesis and transpira­ 107. Schmidt, U. and F. Huber. Methylation of organolead and tion to lead contamination. J. Environ. Qual. lead (II) compounds to (CH3)4Pb by microorganisms. 5(2):156-158, 1974. Nature. 259(5539):!57-158, 1976. 93. Bazzaz, F. A., R. W. Carlson, and G. L. Rolfe. The effect 108. Wood, J. M. Biological cycles for toxic elements in the en­ of heavy metals on plants: Part I. Inhibition of gas ex­ vironment. Sci. 755:1049-1052, 1974. change in sunflower by Pb, Cd, Ni and Ti. Environ. Poll. 109. Wood, J. M. Metabolic Cycles for Toxic Elements in the 7:241-246, 1974. Environment - A Study of Kinetics and Mechanism. In: 94. Wong, D., and Govindjee. Effects of lead ions on photo­ Proc. Inter. Conf. on Heavy Metals in thc Aquatic En­ system I in isolated chloroplasts: Studies on the raction vironment. (P. A. Krenkel, ed.) Pergamon Press, New center P700. Photosynthetica. 70(3):241-254, 1976. York. 1975. p. 105-112. 95. Miller, R. J. and D. E. Koeppe. Accumulation and 110. Wong, P. T. S., Y. K. Chau, and P. L. Luxon. Methylation physiological effects of lead in corn. In: Trace Substances of lead in the environment. Nature. 255(5489):263-264, in Environmental Health. Vol. IV. (D. D. Hemphill, ed.) 1975. University of Missouri Press, Columbia, Mo. 1970. p. 111. Devigne, J. P. Precipitation of lead sulfide by a soil 186-193. micrococcus. C. R. Hebd. Seances Acad. Sci. Ser. D. 96. Bittell, J. E., D. E. Koeppe, and R. J. Miller. Sorption of 267(10):935-937, 1968. heavy metal cations by corn mitochondria and the effects 112. Hass, W. R. and S. Miller. Effects of Various Metals on on electron and energy transfer reactions. Physiol. Plant. Aerobic Organisms. IIT Research Institute, Chicago, Il­ 50:226-230, 1974. linois. Rep. No. IITR1-C82I3-2. 1971. 97. Garber, K. Heavy metals as air pollution - Lead - Zinc - 113. Deason, W. D. Effects of Heavy Metal Ions on Bacteria Cadmium - Influence on vegetation. VDI Ber. 205:50-57, Isolated from a Mine Polluted River. Ph.D. Dissertation. 1975. Colorado State Univ., Ft. Collins. 1970. 98. Ganje, T. J. and A. L. Page. Lead concentrations of plants, 114. Hata, T. In Norinsho Sulsan Koshusko Kenkyo Hokoku. soil, and air near highways. Calif. Agr. 26(4):7-9, 1972. (In Japanese.) 9:363, 1960. 99. Ruhling, A. and G. Tyler. Sorption and retention of heavy 115. Bhuiya, M. R. H. and A. H. Cornfield. Effects of addition metals in the woodland moss, Hylocomiwn Splendens of 1000 ppm CU, NI, Pb, and Zn on carbon dioxide (Hedw.) Br. et Sch. Oikos. 27(0:92-97, 1970. release during incubation of soil alone and after treatment with straw. Environ. Poll. 5:173-177, 1972. 100. Tyler, G. Moss analysis—a method for surveying heavy 116. Cole, M. A. Lead inhibition of enzyme synthesis in soil. metal deposition. In: Proc. 2nd Int'l. Clean Air Congress, Appl. Environ. Microbiol. 55(2):262-268, 1977. Washington, D.C. Academic Press, New York. Paper 117. Watson, A. P., R. I. Van Hook, D. R. Jackson, and D. E. SU-30F. 1971. p. 129-132. Reichle. Impact of a Lead Mining-Smelting Complex on 101. Tyler, G. Heavy metals pollute nature, may reduce pro­ the Forest-Floor Litter Arthropod Fauna in the New ductivity. Ambio. 7(2):52-59, 1972. Lead-Belt Region of Southeast Missouri. Environmental 102. Trollope, D. R. and B. Evans. Concentrations of copper, Sciences Division, Oak Ridge National Laboratory, Oak iron, lead, nickel, and zinc in freshwater algal blooms. En­ Ridge, Tenn. ESD Pub. No. 881, ORNL/NSF/EATC-30. viron. Poll. 77:109-116, 1976. July 1976.

8-16 9. QUANTITATIVE EVALUATION OF LEAD AND BIOCHEMICAL INDICES OF LEAD EXPOSURE IN PHYSIOLOGICAL MEDIA

In this chapter, the state of the art regarding lead workers,4-5 for example, whole blood obtained measurement of lead and biochemical markers of by finger prick is spotted onto Whatman No. 4 lead exposure is summarized, and the accuracy and qualitative paper. Either 9.0-mm or 4.0-mm discs precision of various methods of intra- and inter­ are then punched out and analyzed by the Delves laboratory comparison are examined. The relative cup microtechnique or the carbon-cup flameless diagnostic merits of one type of measurement with atomic absorption procedure. These methods corre­ respect to another — blood lead versus urinary lead, late well with blood obtained by venous puncture. for example — are discussed elsewhere in the docu­ Joselow and Bogden6 have employed a micro- ment. method for routine mass screening of children using a paper disc-in-Delves-cup technique. Capillary 9.1 GENERAL SAMPLING PROCEDURES blood is allowed to drop onto an 8.2-cm disc of FOR LEAD IN BIOLOGICAL MEDIA Schleicher and Schwell No. 903 filter paper so as to The occurrence of lead in physiological media of form a spot somewhat larger than 1/4 in. in interest in this discussion is at the trace level, even diameter, and discs of 1/4-in. diameter are punched under conditions of marked exposure. Therefore, a out directly into previously conditioned Delves number of sample collection and handling precau­ cups. A correlation coefficient of 0.9 was obtained tions are called for, both to minimize loss of lead when results of this method were compared with a from samples and to avoid the contamination of macroprocedure using venous blood and the pro­ samples by this ubiquitously distributed element. cedure of Hessel.7 Sample-gathering problems are of special concern Cook et al.,8 in their investigation of capillary in blood lead screening programs in which great blood collected on paper and of blood volume numbers of samples must be gathered, transported, gathered by venous puncture, obtained a correlation 1 and processed in the shortest possible time. Sample approaching 0.8. collection details for blood lead and other biologi­ Puncture-site preparation is also important in cal indicators of exposure have been reviewed in the sampling. Marcus et al.' report that a preliminary 2 clinical literature. cleaning with ethanolic citric acid solution followed In blood studies it is often desirable to use capil­ by a 70-percent ethanol rinse is satisfactory, whereas lary blood by finger prick instead of venous Cooke et al.8 chose to employ a vigorous scrubbing puncture, especially when young children are in­ with low-lead soap solution and deionized water volved. A number of studies have shown that capill­ rinsing. ary and venous blood are essentially identical in A second precaution with finger-prick sampling is 3 lead content: Mitchell et al. obtained a correlation the way in which the blood flow is expedited for factor of 0.92. However, it should be emphasized sampling. Gravity flow or direct uptake (filter that such results can be achieved only when extreme paper) is preferable to squeezing the finger tip, as care is used in cleaning the skin, and when con­ hard squeezing might dilute the blood drop with tamination of capillary tubes and syringe is avoided. tissue fluid. A number of workers have used filter paper discs Further precautions include the use of for blood sample collection as an alternative to han­ polypropylene syringes9 with needles of stainless dling discrete blood volumes. The paper for this steel and polypropylene hubs for puncture sampling. purpose must be selected for uniformity of manufac­ Urine-sample collection requires acid-washed ture, low lead content, and uniform blood dispersal. plastic containers (and caps) and should include a In the methods of Cernik and Sayers involving low-lead bacteriocide if samples are stored.

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Hard- and soft-tissue gathering from laboratory scribed above mainly in permitting removal of animals or human sources entail surface debride­ bismuth at pH 3.4 as the dithizonate. ment of ambient lead encountered in the process of At present, the colorimetric method has been sampling. Hair cleaning before analysis may be car­ largely supplanted by two other techniques: atomic ried out via the method of Hammer et al.;10 bone absorption (AA) spectrometry in all its variations and teeth may be given a quick rinse in EDTA solu­ and anodic stripping voltammetry (ASV). tion. With soft tissue, the outer layer may be Ofthese two analytical approaches, the more tech­ removed or a segment of underlying matter excised. nically popular, by far, is AA spectrometry, which is For organs of heterogeneous morphology such as used for both macro- and micro-scale analyses. The kidney, it may be more desirable to subject the sam­ theoretical basis for AA and its instrumental design ple to a quick rinse in an ionic chelant, such as are beyond the scope of this presentation; basic 12 EDTA, that does not penetrate beyond the outer reviews are provided by Christian and Feldman 3 membrane. and by L'Vov,' however. Regardless of specific methodology employed, all Macro-scale AA analysis involves direct aspira­ reagents used in lead determinations in biological tion of suitably treated lead-containing samples into media should be certified for low-lead content; and a flame for lead-atom generation and excitation. samples should be stored in a manner that minimizes Micro-AA, which is being used more widely as ac­ lead contamination from air or surfaces. Standard cessories and instrument refinements become com­ lead solutions should be prepared frequently, either mercially available, is of two types: flame and flame­ from stock solutions from analytically certified less, with the latter employing thermoelectric sources or from the pure metal. Solution preparation systems in lieu of a flame. in glass should be minimized, particularly when Of the flame microtechniques for AA analysis of analysis is at the sub-part-per-million level. blood samples, the most widely used is Delves cup procedure14 in which small volumes of blood, 9.2 BLOOD LEAD 10 to 100 fi\, are placed in lead-free nickel cruci­ The first generally accepted practice for measur­ bles. After the organic matrix is destroyed, the cups ing lead in blood and other biological media in­ are inserted into a flame. The Overall configuration volved a spectrophotometric technique based on the of the system permits the optical path to be max­ binding of lead with a chromogenic agent to yield a imally occupied by the lead atom population origi­ chromophoric product. In this connection, the nally present in the sample. Destruction of the classic ligating agent has been dithizone-1, 5- organic matrix in blood may be either partial, using diphenylthiocarbazone. The lead dithizonate is hydrogen peroxide, or total, with pre-ignition ofthe measured spectrophotometrically at 510 nm. organic matter caused by placing the cups near the Two reliable variations ofthe spectrophotometric flame.15 technique when dealing with lead content of 1 to 10 Increasing use is being made of flameless A A, par­ ppm are the USPHS and APHA procedures. ticularly the heated graphite furnace accessory, 9 The USPHS assay is a double-extraction, mixed- whereby volumes of blood are reduced to —- 1 /nl, color procedure having bismuth as the chief inter­ and also whereby in-situ destruction of organic mat­ ferent. Blood (and urine) samples are wet-ashed ter may be achieved. using concentrated nitric acid certified as to low- The electrochemical technique known as ASV16 is lead content. After digest treatment with hydrox­ also* coming into common use in a number of ylamine and sodium citrate, the pH is adjusted to 9 laboratories, particularly for blood and urine lead to 10, and cyanide ion is added. Formation and ex­ analysis. As developed by Matson and Roe,16 the traction of lead dithizonate is carried out using a technique involves concentrating an ion such as chloroform solution of dithizone. Lead is then re-ex­ divalent lead on a negative electrode during a pre­ tracted into dilute nitric acid (1:99 water), and the determined plating time (5 to 60 min) followed by aqueous layer is treated with ammonia-cyanide solu­ polarity reversal and increase for short periods to tion and re-extracted with dithizone-containing yield a discrete current peak that is proportional to chloroform. The organic extracts are read in a ion concentration. spectrophotometer at 510 nm. Although bismuth in­ Also in current use are X-ray fluorescence terferes, this element is encountered infrequently in spectrometry and neutron-activation analysis, two biological media. sophisticated instrumental methods for trace The APHA procedure" varies from that de­ analysis. The considerable expense of the equipment

9-2 and the amount of operator expertise involved 9.5 HAIR LEAD rather limit their use to that of regional service An attractive feature of the clinical use of hair- facilities or central laboratories. The two methods lead levels is its noninvasive nature and the have a distinct advantage, however, in that they per­ feasibility of assembling a rough time frame for lead mit multiple-element analysis, a feature that will be exposure by isolating discrete segments of the total of increasing importance as more is unveiled about hair length. the complex interactions oflead with other metals in A serious drawback in hair analysis, however, is man and other organisms. the level of contamination by ambient air lead, lead in hair preparations, etc., as discussed by Hammer et 9.3 URINE LEAD al.'°Hair measurements without prior treatment of Precautions to be taken for urine sample collec­ the sample include both exogenous and endogenous tion were noted earlier. Because ofthe considerable sources. Examples ofthe former are dyes, shampoos, amount of ionic matter in urine, it is usually necess­ sweat, and dust. Pre-analysis hair washing with ary to manipulate urine samples in various ways detergent and EDTA removes most ofthe externally before analysis. found lead; but there is still no definitive way to All of the methods employed for blood lead determine whether any cleaning technique removes analysis as described above may also be applied to the contamination portion of lead and leaves the in­ assessment of urine lead levels. Care, however, ternal lead content undisturbed. should be exercised in the analysis of urine samples Hair is usually wet-ashed before analysis, and the from patients undergoing chelation therapy, with digest is diluted and analyzed by AA spectrometry. special attention to how a specific procedure will ac­ Because relatively high levels of lead are encoun­ commodate or be interfered with by lead excreted as tered in hair, small sample sizes can be used when a a complex, e.g., lead-EDTA. Prior ashing of urine sensitive procedure such as AA spectrometry is samples will minimize complications in this regard, employed. but partial degradation or no prior treatment might necessitate co-analysis of lead in the complex form for standards. 9.6 LEAD IN TEETH AND BONE 9.4 SOFT-TISSUE LEAD The biochemical significance of lead levels in teeth and bone is discussed elsewhere. From an Because ofthe nature of this medium, it is usually analytical standpoint, bone samples are usually ob­ necessary either to ash or to solubilize tissue sam­ tained from experimental animal studies. Bone sam­ ples. Wet-ashing is rapid and avoids lead loss via ples must first be debrided of muscle and connective volatilization, but it requires use of corrosive acids tissue and chemically rid of surface lead contamina­ and procedural care to avoid contamination of tion by rinsing with EDTA or other chelant solution. reagents and other problems. Dry ashing, on the other hand, is simple and uses no contaminating Bone and teeth are usually wet- or dry-ashed reagents. The drawbacks are mainly those of lead before analysis, and the relative merits of these volatilization and retention ofthe element in refrac­ mineralizing procedures are as noted above with soft tory residues. Newer techniques of ashing include tissue assays. Because ofthe high mineral content of (1) low-temperature ashing, in which dry samples bone and teeth, care must be taken to avoid are mineralized in an evacuated chamber that is spectrochemical interference by calcium, phosphate, bathed in an energy-rich plasma via r-f discharge in etc. The relatively high levels of lead in these two an oxygen stream, and (2) use of the combustion hard tissues, however, permit dilution ofthe samples bomb, in which samples are heated in acid an at ele­ for testing. Because the effect of the high mineral vated temperature in a sealed inert vessel. content on analytical signals is probably sufficient to A newer method of tissue handling, solubilization, preclude the use of simple aqueous lead standards, it entails the treatment of samples with quaternary am­ is advisable to employ workup solutions of bone monium compounds and analysis of aliquots, chiefly samples that have first been analyzed for lead con­ by AA spectrometry.17 tent and to which known amounts of lead from a The bulk of the current literature centers on the stock solution are then added. Matrix standards pre­ use of atomic absorption spectrometry as the method pared in this fashion are assumed to reflect the in­ of choice for assessing lead levels in soft tissue. fluence of mineral content on all of the samples.

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9.7 COMPARATIVE STUDIES OF METHODS by Pierce et al.2 These same authors describe the FOR MEASUREMENT OF LEAD IN BIOLOGI­ state ofthe art critically and offer some recommen­ CAL MEDIA dations for improving the quality of blood lead In an interlaboratory study of the USPHS col­ analyses: orimetric method for lead in blood and urine, 1. Every laboratory should have established Keenan et al.18 reported the results from 10 partici­ quality-control procedures. pating laboratories. For blood, a mean lead value of 2. Control procedures should include replicate 26 ± 0.82 /ig/dl was obtained; spiked samples gave analyses, recovery of known additions or virtually identical correspondence among the spikes, participation in interlaboratory tests, groups. Urine samples with lead added gave values and analyses of known materials. from the reporting groups with a mean of 679 ± 5.5 3. Testing samples that the analysts analyze /xg/liter. blind should be used to minimize bias. 28 Microscale A A techniques for whole-blood lead It has been suggested that the acceptable agree­ have been found to show good correspondence with ments in blood lead levels found when a single results obtained using conventional flame pro­ laboratory employs different techniques compared cedures.19-20 with the wide variance found when different Matson21 has reported good correlations for lead laboratories employ different methods on portions levels in blood and urine when comparing ASV with of common blood samples relate primarily to pre­ a colorimetric and an AA procedure. Similarly, paration and state of the blood samples before and during distribution and subsequent analysis. The Horiuchi et al.22 saw little difference in lead levels 29 for blood and urine when contrasting ASV, A A, and results of Grimes and coworkers bear this out polarography. because a large number of carefully controlled blood samples analyzed by their laboratories, using Interlaboratory studies of various methods for the paper disc technique, provided results that com­ lead analysis of biological media have yielded some­ pared well with those of other laboratories using what disappointing results.23*25 In a recent study in­ other instrumentation with the disc technique. volving 66 laboratories throughout Europe, blood and urine determination variance was observed to be 9.8 MEASUREMENT OF URINARY 25 unacceptably high. 8-AMINOLEVULINIC ACID (ALA-U) Presently, the Center for Disease Control (CDC) Some comments regarding sample collection for is carrying on a monthly proficiency testing program ALA-U are necessary. ALA is stable in acidified for blood lead involving approximately 200 urine (pH 1 to 5), so that acetic or hydrochloric acid laboratories. Results are made available through addition is satisfactory at the time of sample collec­ monthly reports for analysis of bovine blood sam­ tion. If samples are stored in the dark at 4°C, the ples.26 The criterion used by CDC for assessing un­ ALA content remains relatively constant for several satisfactory performance is: greater than 15 percent months.30 relative deviation at levels of 40 figld\ or above and ALA-U measurement usually entails the classic greater than 6 Mg/dl at lower levels. 31 In a recent CDC report (Survey 1977I)27 covering method of Mauzerall and Granick. In this ap­ 130 laboratories for testing and 26 reference proach, ALA-U is condensed with a /3-dicarbonyl laboratories using three bovine blood samples (cows compound such as acetylacetone or ethyl acetoace- fed lead acetate), 72 percent of the tested laborato­ tate to yield a substituted pyrrole derivative; this in­ ries were within the acceptable range for a sample termediate is then caused to react with Ehrlich having a mean of 16.6/u.g/dl. The acceptable percen­ reagent (p-dimethylaminobenzaldehyde) to yield an tage was lower, interestingly, at higher sample intense ionic chromophore. This procedure is not means of 48.2 and 54.6 /ug/dl (64 and 67 percent, specific for ALA-U, however, since aminoacetone respectively). When results were tabulated as a func­ interferes. Though the significance of such inter­ tion of method, the Delves cup A A spectrometric ference is marginal when ALA-U levels are and ASV methods furnished the smallest coefficients markedly elevated, it becomes very important when of variation. only slight elevations of ALA-U are being measured. A number of other proficiency programs are pre­ First, urine samples are chromatographed on ion- sently operating in the United States, and the results exchange resin columns (Dowex-2), the ALA-U of these have been included, along with the Euro­ being co-eluted with urea using water as eluent. pean program, in a recently published monograph Eluate transfer to a Dowex-50 column is followed

9-4 P.30

by sequential elution with water to remove the urea is not advisable because competition for lead may and then with acetate solution to permit removal of reactivate the lead-inhibited enzyme. the ALA-U. Treatment with acetylacetone and heat­ Minimal time lapse should occur between collec­ ing to effect complete condensation is followed by tion and enzyme assay—no more than 24 hr if sam­ treatment of sample aliquots with modified Ehrlich ples of heparinized blood are held at 4°C. reagent (p-dimethylaminobenzaldehyde in per­ The chemical basis for measuring enzyme activity chloric/acetic acid). The resulting chromophoric salt involves spectral measurement of the amount of is allowed to achieve maximum intensity (ca. 15 porphobilinogen generated from ALA, the porpho­ min) after which the sample is read in a spectro­ bilinogen being condensed with p-dimethyl­ photometer at 553 nm. The detection limit is 3 aminobenzaldehyde to yield a chromophore that is ^moles/liter urine, and chromophore stability is measured at 553 nm. Mercury (II) is employed to limited to about 15 min. minimize the interference effect of sulfhydryl en­ A number of modifications to the above basic ap­ tities present in the medium. proach have been reported. Several reports attempt The micromethod of Granick and co-workers40 to take into account the interference posed by requires only 5 fi 1 of whole blood and appears to be aminoacetone. The quantitative corrections to be of value in a screening program. Enzyme incubation used are described by the authors of these re­ is done at 37°C for about 60 min; ALA ofthe highest ports.32-33 possible purity is necessary as a substrate. The initial isolation of porphobilinogen is omitted Termination of the reaction (enzyme activity) is (in cases where porphyria is not suspected) in the done via trichloroacetic acid. modification of Williams and Few,34 in which a cor­ The activity of the enzyme may be calculated in relation of 0.99 was observed with the reference two ways:40-41 technique using samples from 39 lead workers. Doss Activity = AOD553(sample-tissue control) and Schmidt35 report that use of commercially avail­ 138,000 nMol PBG/ml RBC/hr able dual ion-exchange columns offers results that HCI compare favorably with the Mauzerall and Granick 00 = AOD5„(t60'-t0')x method.31 HCL The assay has been automated with good corres­ x 131.48 nMol PBG/ml RBC/min pondence of results to the manual method in the In the European standardized method for ALA 36 37 laboratories of Grisler et al. and Lauwerys et al. determination42 aimed specificially at enzyme levels In another variation ofthe ALA-U procedure, the in blood corresponding to low levels of exposure to 38 method of Schlenker et al. removes aminoacetone environmental lead, incubation of the enzyme in which interferes with the assay. In the procedure of three aliquots of blood (0.2 ml cooled to 4°C) is car­ MacGee et al.,39 urinary and blood ALA is deter­ ried out in the presence of excess 8-aminolevulinic mined by gas-liquid chromatography, a highly acid. An aliquot blank is also carried through the specific technique. procedure. Hemolysis of the cells and incubation with substrate is followed by quenching with mer­ curic chloride-trichloroacetic acid solution. 9 9 MEASUREMENT OF 8-AMINO­ Centrifugation and treatment with modified LEVULINIC ACID DEHYDRATASE (ALA-D) Ehrlich's reagent is followed at 5 min by absorbance Located in erythrocytes, 8-aminolevulinic acid measurement. Blood samples are preferably run dehydratase (ALA-D) catalyzes the conversion of within 3 hr and in no case after 24 hr when held at ALA to porphobilinogen in the heme biosynthetic 4°C. pathway. Its inhibition by heavy metals such as lead 43 indicates that it is a sulfhydryl enzyme and also In a study by Granick et al. the activity of ALA- forms the biochemical basis for assessing its activity D before and after treatment with dithiothreotol in lead-exposed organisms. (DTT) is determined. The DTT (added in vitro, 20 mM) provides -SH groups and reactivates the Blood collection requires the use of low-lead enzyme completely at all concentrations of blood tubes containing anticoagulant, whereas for lead. Because DTT-reactivated ALA-D yields total micromeasurement, blood is collected in a enzyme activity, variation in levels of the unacti- heparinized microhematocrit tube.40 Obviously, use vated enzyme may be normalized by determination of strong chelants, such as EDTA, as anticoagulants ofthe rates of both activities. Hence a person having

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a high ALA-D for genetic reasons at a given blood those noted above, but it is varied to employ 1.5N level of lead will have relatively high activity for HCI to generate the fluorescing acid layer. both activated and nonactivated enzyme, and the ac­ Measurement is at 610 nm and excitation at 405 nm tivity ratio will depend less on genetic factors than with coproporphyrin employed as the standard. on lead inhibition. Consequently, correlation is Observing that FEP is actually the zinc complex markedly improved. This study also shows that in­ (ZPP), Lamola and coworkers49 have devised a hibition by lead is of the noncompetitive type. rather rapid and sensitive fluorometric procedure in which 20 fi\ of whole blood is worked up in a 9.10 MEASUREMENT OF FREE ERYTHRO­ detergent-phosphate buffer solution (dimethyl- CYTE PROTOPORPHYRIN (FEP) dodecylamine oxide) and fluorescence measured at Another reaction that lead inhibits in the human 594 nm with excitation at 424 nm. heme biosynthetic pathway is heme formation. As a In the procedure of Chisolm and Brown,50 which result of blocking this reaction, porphyrins, particu­ has been evaluated as a selected method by Schwartz larly protoporphyrin IX (actually zinc-pro- and Piomelli, 2Q-fi\ blood volumes are treated with toporphyrin), accumulate in the erythrocytes. ethyl acetate/acetic acid solution (3:1) and agitated Measurement of protoporphyrin IX specifically, or for 30 sec. After centrifugation, the layers are ex­ all erythrocyte prophyrins together, is generally tracted with 3N HCI, and the acid layer is diluted referred to as the free erythrocyte protoporphyrin with more 3N HCI. The acid extracts are analyzed (FEP) test. spectrofluorometrically with coproporphyrin em­ The spectrochemical properties of FEP that form ployed as the quantitating standard. the basis for its measurement include its lability to A portable hematofluorometer that utilizes front- light and strong acids, its metal coordinating ability, face optics, internal standards, and built-in com­ its possession of an absorption spectrum in the Soret putational capabilities permits the assessment of band region, and its marked intensity of fluores­ erythrocyte zinc protoporphyrin (ZPP). As cence. Spectral methods used, however, must take developed by Bell laboratories51 and subsequently into account the fact that copro- and uroporphyrin made available commercially,52 the apparatus per­ provide considerable interference in FEP measure­ mits the analysis of a drop of blood applied to a ment. Although both absorption spectrophotometric cover slip directly from finger pricking. The ZPP and fluorometric methods may be employed for FEP level {fig ZPP/dl blood) is automatically calculated 4446 assessment, fluorometric techniques carried out and displayed on a digital readout. on a microscale are more frequently used because of A number of micromethods for FEP analysis have the relatively cumbersome nature of absorption been critically evaluated by Hanna et al. 53: Double spectrometry in terms of time and materials. These extraction with ethyl acetate/acetic acid-HCl,48 microfluorometric techniques, in particular, provide single extraction with ethanol, single extraction with a rapid, relatively accurate means of screening acetone,54 and direct solubilization with detergent pediatric populations. buffer.49 Of these, the ethyl acetate and ethanol pro­ 47 In the microtechnique of Granick et al. , several cedures were satisfactory; the complete extraction of microliters of whole blood are placed in 1-ml test FEP makes the former the technique of choice when tubes that also serve as fluorometric cuvettes. Addi­ an absolute value rather than technical simplicity is tion of ethyl acetate/glacial acetic acid (2:1) is then of primary concern. rapidly followed by treatment with 0.5N HCI and vigorous shaking. The acidic phase (bottom layer) 9.11 REFERENCES FOR CHAPTER 9 contains the bulk ofthe porphyrins. The cuvettes are scanned over the range 560 to 680 nm, using excita­ 1. Marcus, M., M. Hollander, R. E. Lucas, and N. C. Pfeiffer. tion at 400 nm. The ratio ofthe two-band maxima at Micro-scale blood lead determinations in screening: Evaluation of factors affecting results. Clin. Chem. 605 and 655 nm is measured for each sample with a 2/(4):533-536, 1975. ratio of 2:1 indicating only FEP. Any lesser value in­ 2. Pierce, J. O., S. R. Koirtyohann, T. E. Clevenger, and F. E. dicates copro- and/or uroporphyrins. In the latter Lichte. The Determination of Lead in Blood. Interna­ case, an extraction with 0.05N HCI on analysis ofa tional Lead Zinc Research Organization, Inc., New York. second sample removes copro- and uroporphyrin. 1976. 48 3. Mitchell, D. C, K. M. Aldous, and F. J. Ryan. Mass The technique of Piomelli, using 20 fi\ of blood screening for lead poisoning: Capillary blood sampling added to a 5-percent Celite suspension in saline, uses and automated Delves-cup atomic-absorption analysis. essentially the same initial extraction procedures as N.Y. State J. Med. 74:1599-1603, Aug. 1974.

9-6 4. Cernik, A. A. arid M. P. H. Sayers. Determination of lead 22. Horiuchi, K., S. Horiguchi, F. Takoda, and K. Teramoto. in capillary blood using a paper punched disc atomic ab­ A polarographic method for the determination of a small sorption technique. Applications to the supervision of lead amount of lead in biological materials. Osaka Med. J. workers. Brit. J. Ind. Med. 28:392-398, 1971. /4:II3-I18, 1968. 5. Cernik, A. A. Determination of blood lead using a 4.0 mm 23. Keppler, J. F.. M. E. Maxfield. W. D. Moss, G. Tietjen. paper punched disc carbon cup sampling technique. Brit. and A. L. Linch. Interlaboratory evaluation of the J. Ind. Med. J/:239-244, 1974. reliability of blood lead analyses. J. Am. Ind. Hyg. Assoc. 6. Joselow, M. M. and J. D. Bogden. A simplified micro 57:412-429, 1970. method for collection and determination oflead in blood 24. Donovan, D. T., V. M. Vought, and A. B. Rakow. using a paper disk-in-Delves cup technique. At. Absorp. Laboratories which conduct lead analysis on biologic Newsletter. 77:99-101, 1972. specimens. Arch. Environ. Health. 2J(2):1 11-113, 1971. 7. Hessel, D. W. A simple and rapid quantitative determina­ 25. Berlin, A.. P. Del Castilho, and J. Smeets. European inter tion oflead in blood. At. Absorp. Newsletter. 7:55, 1968. comparison programmes. In: Environmental Health 8. Cooke, R. E., K. L. Glynn, W. W. Ullmann, N. Lurie, and Aspects of Lead. Commission of the European Com­ M. Lepow. Comparative study of a micro-scale test for munities, Centre for Information and Documentation, lead in blood, for use in mass screening programs. Clin. Luxembourg. May 1973. p. 1033-1049. Chem. 20(51:582-585, 1974. 26. Blood Lead Proficiency Testing. U.S. Department of 9. Lead: Airborne Lead in Perspective. National Academy of Health, Education, and Welfare, Center for Disease Con­ Sciences, Washington, DC. 1972. trol, Atlanta. Ga. 1975. 10. Hammer, D. I.. J. F. Finklea, R. H. Hendricks. C. M. Shy. 27. Blood Lead Proficiency Testing Program. U.S. Depart­ and R. J. M. Horton. Trace-metal concentrations in ment of Health, Education, and Welfare, Center for Dis­ human hair. In: Helena Valley, Montana. Area Environ­ ease Control, Atlanta, Ga. Monthly Report for March, mental Pollution Study. U.S. Environmental Protection 1977. Agency. Research Triangle Park, N.C. Pub. No. AP-91. 28. Lead: Environmental Health Criteria 3. World Health 1972. p. 125-134. Organization and the United Nations Environment Pro­ 11. Methods for Determining Lead in Air and Biological gramme, Geneva. 1977. p. 59-65. Materials. American Public Health Association, New 29. Grimes, H.. M. P. H. Sayers, A. A. Cernik, A. Berlin, P. York. 1955. 69 p. ' Recht, and J. Smeets. Note on the Lead Exposure of 12. Christian, G. D. and F. J. Feldman. Atomic Absorption Children: Determinations Carried Out on Behalf of the Spectroscopy: Applications in Agriculture, Biology and Commission in Western Ireland. Commission ofthe Euro­ Medicine. Wiley-Interscience, New York. 1970. pean Communities, Luxembourg. Report No. VF-I49I. 13. L'Vov, B. V. Atomic Absorption: Spectrochemical 1975. p. 7. Analysis. J. H. Dixon (tr). American Elsevier Publishing 30. Haeger-Aronsen, B. Studies on urinary excretion of 8- Co., New York. 1971. aminolevulinic acid and other haem precursors in lead 14. Delves, H. T. A micro-sampling method for the rapid workers and lead-intoxicated rabbits. Scand. J. Clin. Lab. determination of lead in blood by atomic-absorption Invest. /2(Suppl. 47>—1-128. 1960. spectrophotometry. Analyst. (London) 95:431-438, May 31. Mauzerall. D. and S. Granick. The occurrence and deter­ 1970. mination of 8-aminolevulinic acid and porphobilinogen in 15. Ediger, R. D. and R. L. Coleman. A modified Delves cup urine. J. BioTchem. 2/9(0:435-446, 1956. atomic absorption procedure for the determination of lead 32. Marver. H. S.. D. P. Tschudy. M. G. Perlroth. A. Colline, in blood. At. Abs. Newsletter. 7/(2):33-36, 1972. and G. Hunter. Jr. The determination of aminoketones in 16. Matson, W. R. and D. K. Roe. Trace metal analyses of biological fluids. Anal. Biochem. /4.-53-60. 1966. natural media by anodic stripping voltammetry. Anal. In- 33. Urata. G. and S. Granick. Biosynthesis of 8-aminoketones strum. 4:l'9-22, 1966. and the metabolism of aminoacetone. J. Biol. Chem. 1 7. Murthy, L., E. E. Menden, P. M. Eller, and H. G. Petering. 258(2):8ll-820. 1963. Atomic absorption determination of zinc, copper, cad­ 34. Williams, M. K. and J. D. Few. A simplified procedure for mium and lead in tissues solubilized by aqueous the determination of urinary 8-aminolevulnic acid. tetramethylammonium hydroxide. Anal. Biochem. Brit. J. Ind. Med. 24:294-296, Oct. 1967. 55(2):365-372, 1973. 35. Doss. M. and A. Schmidt. Quantitative determination of 18. Keenan.R.G., D.H. Byers, B. E. Saltzman, and F. L. Hy- 8-aminolevulinic acid and porphobilinogen in urine with slop. The "USPHS" method for determining lead in air ready-made ion-exchange chromatographic columns. Z. and in biological materials. J. Am. Ind. Hyg. Assoc. Klin. Chem. Klin. Biochem. 9:99-102, 1971. 24(5 ):481-491, 1963. 36. Grisler, R.. M. Genchi, and M. Perini. Determination of 19. Kubasik, N. P., M. T. Volosin, and M.H. Murray. Carbon urinary 8-aminolevulinic acid by continuous flux and se­ rod atomizer applied to measurement of lead in whole quential automatic analyzers. Med. Lav. 60:678-686, Nov. blood by atomic absorption spectrophotometry. Clin. 1969. Chem. 7S(5):4I0^I2, 1972. 37. Lauwerys. R.. R. Delbroeck, and M. D. Vens. Automated analysis of 8-aminolevulinic acid in urine. Clin. Chim. 20. Hicks, J. M., A. N. Gutierrez, and B. E. Worthy. Evalua­ Acta. 40:443-447. 1972. tion of the Delves micro system for blood lead analysis. 38. Schlenker, F. S., N. A. Taylor, and B. P. Kiehn. The Clin. Chem. 79(3):322-325, 1973. chromatographic separation, determination, and daily ex­ 21. Matson, W. R. Rapid sub-nanogram simultaneous cretion of urinary porphobilinogen, aminoacetone, and analyses. Trace Substances in Environ. Health. delta-aminolevulinic acid. Am. J. Clin. Pathol. IV:396-406, 1970. 42:349-354. 1964.

9-7 Hr***"

39. MacGee, J., S. M. Roda. S. V. Elias, A. Lington, M. W. samples of whole blood: Applications to metabolic defects Tabor, and P. B. Hammond. Determination of delta- involving the heme pathway. Proc. Natl. Acad. Sci., U.S.A. aminolevulinic acid in blood plasma and urine by gas- 69:2381-2385, 1972. liquid chromatography. Biochem. Med. 77:31-44, 1977. 48. Piomelli, S. A micro-method for free erythrocyte 40. Granick, J. L., S. Sassa. S. Granick, R. D. Levere, and A. porphyrins: The FEP test. J. Lab. Clin. Med. 87:932-940, Kappas. Studies of lead poisoning. I. Microanalysis of 1973. erythrocyte protoporphyrin levels by spectrofluorometry 49. Lamola, A. A., M. Joselow, and T. Yamane. Zinc pro­ in the detection of chronic lead intoxication in the toporphyrin (ZPP): A simple, sensitive, fluorometric subclinical range. Biochem. Med. 8:135-148, 1973. screening test for lead poisoning. Clin. Chem. 41. Weissberg, J. B., F. Lipschutz, and F. A. Oski. 8- 27(l):93-97, 1975. aminolevulinic acid dehydratase activity in circulatory 50. Chisolm, J. J., Jr., and D. H. Brown. Micro-scale photo- blood cells: A sensitive laboratory test for the detection of fluorometric determination of "free erythrocyte childhood led poisoning. New Eng. J. Med. porphyrin" (protoporphyrin IX). Clin. Chem. 284(ll):565-569. 1971. 27(11):1669-I682, 1975. 42. Berlin. A. and K. H. Schaller. European standardized 51. Blumberg, W. E., J. Eisinger. A. A. Lamola, and D. M. method for the determination of 8-aminolevulinic acid Zuckerman. The hematofluorometer. Clin. Chem. dehydratese activity in blood. Z. Klin. Chem. Klin. 2J(2):270-274, 1974. Biochem. 72:389-390, 1974. 52. Matson, W. R., R. M. Griffin, T. J. Sapienza, J. M. 43. Granick, J. L.. S. Sassa. S. Granick. R. D. Levere. and A. Shiavone, and A. J. Reed. A Critical Evaluation of the Kappas. Studies in lead poisoning. II. Correlation between Technical and Operational Utility of Zinc Protoporphyrin the ratio of activated and inactivated 8-aminolevulinic (ZnP), Erythrocyte Protoporphyrin (EP) and Blood Lead acid dehydratase of whole blood and the blood lead level. (BL) in Pediatric Screenings for Lead Insult. Paper pre­ Biochem. Med. 8:149-159. 1973. sented at Region IV DHEW CDC Conference on 44. Wranne, L. Free erythrocytes copro- and protoporphyrin: Pediatric Lead Poisoning Control Projects, Atlanta, Ga. A methodological and clinical study. Acta Pediatrica. February 1976. 49(Suppl. 124):l-78. I960. 53. Hanna. T. L.. D. W. Dietzler. C. H. Smith. S. Gupta, and 45. Heilmeyer. L. Uber die erythropoetischen porphyrien. H. S. Zarkowsky. Erythrocyte porphyrin analysis in the Wien Med. Wochschr. / 76:12-17. 1966. detection oflead poisoning in children: Evaluation of four 46. Langer, E. E.. R. G. Haining. R. F. Labbe. P. Jacoby. E. F. micro methods. Clin. Chem. 22: 161-168. 1976. Crosby, and C. A. Finch. Erythrocyte protoporphyrin. Blood. 40:112-128. 1972. 54. Chisholm, J. J., C. W. Hastings, and D. K. K. Chering. 47. Granick, S.. S. Sassa. J. L. Granick. R. D. Levere. and A. Micro-photo fluorometric assay for protoporphyrin in Kappas. Assays for porphyrins. 8-aminolevulinic-acid acidified acetone extracts of whole blood. Biochem. Med. dehydratase, and porphyrinogen synthetase in microliter 9:113, 1974.

9-8 10. METABOLISM OF LEAD

10.1 INTRODUCTION 10.2.1 Respiratory Absorption The metabolism of lead in man may be defined as The International Radiological Protection Com­ the physiological processes relating to absorption, mission (IRPC) Task Group on Lung Dynamics' distribution, translocation, and net retention. The developed a model designed to predict the percen­ metabolism of lead in man is discussed in this tage of inhaled aerosols that would be deposited and chapter in terms of routes of exposure and of the retained in the lungs. This model predicted that ap­ physiological distinctions, existing within popula­ proximately 35 percent of the lead inhaled in tion classes, that modify metabolic processes, general ambient air would be deposited in the air­ especially with reference to children versus adults. ways. Since the aerodynamic diameter of lead parti­ Most of the material discussed in the following cles is generally in the range of 0.1 to 1.0 fim, pages addresses the dietary habits of adults in the deposition would occur predominantly in the deeper United States. For children, however, there are die­ regions of the lung. Emissions from stationary tary habits that are distinct from those of adults and sources frequently include a significant proportion that have implications for differences in exposure of larger particles that, when inhaled, would be between adults and children. deposited primarily in the nasopharynx. Because these particles usually fall out of the air rather For example, in assessing the indirect contribution quickly, exposure to these larger particles is limited of airborne lead to diet, one must consider the hand- primarily to the near vicinity ofthe emission source. to-mouth activity of young children, i.e., sucking of The deposition within the respiratory tract of very dirty fingers in contact with environmental dust and small particles (<0.1 fim) apparently occurs chiefly dirt; retrieving foodstuffs, such as lollipops, that fall by diffusion,2 and rates or sites cannot be predicted. into dirt; and a host of other childhood activities by The IRPC model predicts a total airway deposition which airborne lead may contribute to the intake of of 40 to 50 percent for 0.5 /xm particles, but a study lead by children but not by adults. in human volunteers indicated only 6 to 16 percent In this chapter, the physiological processes that desposition, depending on the rate and depth of control the uptake of lead by man (absorption) will respiration.3 Chemical composition also affects up­ be discussed first. Then the movement of lead take as does the aging of the aerosol (Chapter 6). through the body into its depot tissues (distribution) This illustrates the difficulty in choosing the ap­ and its eventual elimination (excretion) will be propriate chemical composition4 for studies on treated. deposition.

10.2 ABSORPTION Airway clearance of lead aerosols as predicted by the IRPC lung model' is even more tenuous than are The quantities of lead absorbed from environmen­ predictions regarding deposition. The model indi­ tal sources are determined not only by the amount cates that the absorption or clearance of lead ingested or inhaled but also by the particle size and deposited in the airways would vary greatly depend­ chemical species involved. ing on the solubility and on the inherent toxicity of Absorption depends also on specific host factors the particles to the clearance mechanism (lung such, as age, nutrition, and physiological status. In macrophages and cilia). addition, the total quantity ingested in food and water varies greatly from individual to individual, 10.2.1.1 HUMAN STUDIES and the total quantity inhaled depends on the size Actual studies on the fractional deposition of par­ and weight of the individual and on the energy ex­ ticles in the respiratory tract of man have not been pended in day-to-day activity. extensive, especially in the case of lead. Using an air

10-1 P.35

lead level of 150 /xg/m3, Kehoe57 studied the calculated that under conditions of chronic airborne deposition of combusted tetraethyl lead in human lead exposure roughly 50 percent of the deposited volunteers. The source of lead was combusted lead is absorbed. Although alveolar macrophages tetraethyl lead, which produced lead (III) oxide ingest particles deposited in the lungs, these cells 12 (Pb203) in the air. Subjects breathed air containing may be damaged by inorganic lead compounds. 150 /j.g/m3 lead; the smaller particles averaged 0.05 Such damage has been demonstrated in rats and /um in diameter and the larger ones averaged 0.9 guinea pigs. It is possible, then, that lung defense fim in diameter, as viewed under the electron mechanisms may be impaired when air contains high microscope. This represents a mass median lead concentrations. equivalent diameter of approximately 0.26 and 2.9 8 fim, respectively. Thirty-six percent ofthe smaller 10.2.1.2 ANIMAL STUDIES particles and 46 percent of the larger particles were Animal studies by Bingham et al.13 have demon­ deposited. strated a pronounced reduction in the number of Nozaki9 reported that when lead fumes generated lung macrophages resulting from inhalation of lead in a high-frequency induction furnace were inhaled oxide at both 10 and 150 figlm*. Similar results at a concentration of 10,000 figlm3 deposition was have been reported by others. 12-14>15 This suggests related to respiration rate and particle size. At 10 that the lung clearance mechanism may function less respirations per minute, the deposition decreased effectively when air lead concentrations are high. from 63 to 42 percent as the particle size was Thus, Pott and Brockhaus16 reported that large reduced from 1.0 to 0.05 fim. At 30 respirations per doses of lead bromide solution or lead oxide suspen­ minute, deposition rates were about halved. The sion administered intratracheally to rats (1.5 mg of results, which are similar to those of Kehoe,57 are lead oxide per dose on 8 successive days) were re­ fairly consistent with the IRPC lung deposition tained by the body as completely as were intra­ model.1 venous doses. At one-third the dose, however, reten­ These data suggest that a 30 ± 10-percent deposi­ tion via the intratracheal route was significantly less. tion rate can be expected in individuals breathing Randall and his coworkers17 exposed 4 baboons ambient air and that deposition may vary considera­ to aerosolized lead (Pb304) of varying particle size bly, depending on the particle size and the frequency (mass median diameters of 5.9, 3.2, and 2.0 fim, of respiration. respectively). The air lead concentrations varied The rate of lung clearance has been studied by from approximately 1 to 4 /xg/m3. The exposure means of gamma-ray lung scans following inhalation period lasted 4 weeks, and blood sampling con­ of 212Pb, but the relevance of the results to the rate of tinued for 6 weeks. The rate of absorption of lead clearance ofthe chemical and physical forms of lead into blood was faster and reached a higher level for usually inhaled by man is highly questionable.10 coarse (mean diameter = 1.6 fim) particles than for These studies involved the absorption of 212Pb atoms fine (mean diameter = 0.8 fim). on carrier aerosol particles; however, desorption under these artificial circumstances may be totally 10.2.2 Gastrointestinal Absorption unlike the clearance rate for ambient air lead parti­ cles. 10.2.2.1 HUMAN STUDIES Kehoe57 reported a substantial increase in fecal It must be noted at the outset that the absorption excretion when large-particle lead oxide aerosols of lead from food varies with the physical form of were inhaled for many weeks at 105 /xg/m3; the in­ dietary intake. For example, the literature indicates crease probably resulted from the swallowing of par­ that the percent absorption of lead from beverages is ticles trapped in the nasopharynx. When air with a about five to eight times greater than that from solid similar lead concentration in small particles was in­ food.1820 Kehoe57 concluded from long-term bal­ haled, only a small rise in fecal lead excretion was ance studies that approximately 10 percent ofthe in­ observed. take of lead from food and beverages was absorbed In a recent study, Chamberlain et al." found a 35- from the gastrointestinal tract since this was the percent rate of deposition, at a respiration rate of 15 amount excreted in the urine. This estimate, how­ per minute, when subjects inhaled automobile ex­ ever, disregarded the urinary lead that might have haust fumes containing radioactively labeled come from inhalation, as well as the lead excreted in tetraethyl lead (203Pb). This compares favorably feces after absorption from the gastrointestinal tract. with Nozaki's previous findings.9 These authors" Rabinowitz et al.,21 however, obtained similar

10-2 results using orally administered 204Pb incorporated reaching a subject via a distinctly different route of into the diet. exposure than inhaled lead. Inhalation of lead may Alexander et al.22 studied the absorption of lead be regarded as direct exposure to airborne lead. from the gastrointestinal tract in 8 infants and young Some portion of dietary lead may also be attributed children aged 3 months to 8.5 years and concluded to exposure to airborne lead, but indirect rather than that 53 percent of ingested lead was absorbed. Ab­ direct, with lead reaching food either by deposition sorption and retention were consistent within the age onto aerial edibles or by fallout onto soil and subse­ range studied. This study, however, has been criti­ quent absorption by root crops. (Internal trans­ cized because the values varied greatly. location of lead between roots and aerial parts is ap­ In a recent study, Ziegler et al.23 showed that a parently small.) In addition, variable fractions of in­ greater percentage of intake lead was absorbed and haled lead are ingested after deposition in the air­ retained by infants than by older subjects. In this re­ ways; they are cleared by retrograde movement to port, 2 separate series of investigations were con­ the pharynx, where the particles are then swallowed. ducted. In the first, 3 to 8 balance studies were per­ formed with 9 infants each. In the second, each of 6 Section 7.4.1 cites lead concentrations typically infants consumed randomly allocated diets provid­ found in various foods, but no research has been ing low, intermediate, and moderate amounts of brought to light that clearly partitions the origins of lead. When intakes of lead exceeded 5 /ug/kg/day, food lead. There would seem to be three principal which is a reasonable level given typical dietary pat­ candidates: (1) deposition of airborne lead (on pri­ terns, net absorption averaged 42 percent and reten­ mary food crops and on animal feed crops); (2) ab­ tion averaged 32 percent of intake. It should also be sorption of soil lead (much of this lead is often the noted that there was an inverse relationship between historical accumulation of airborne fallout); and (3) calcium intake and blood lead level. lead acquired in the processing and canning of foods. These results are in general agreement with animal studies and to some extent corroborate the Based on the contrasts between fresh and pro­ findings of Alexander et al.22The study of Ziegler et cessed foods (excluding frozen foods), it would ap­ al.23 appears to be much better designed than that of pear that processing and canning of certain foods Alexander et al.22 regularly doubles or triples their average lead con­ Ingested or dietary lead is often thought of as centrations (Table 10-1).

TABLE 10-1. LEAD CONTENT OF FRESH, PROCESSED, AND CANNED FOODSTUFFS24

Lead Lead Food concentration (ppm) Food concentration (ppm) Fresh produce Canned vegetables Carrots 0.205 Beets 0.381 Lettuce 0.130 Beans 0.318 Potatoes 0.050 Peas 0.425 Avg. 0.128 Tomatoes 0.710 Avg. 0.458 Processed foods Canned juices White flour 0.052 Tomato 0.338 Cornmeal 0.143 Vegetable 0.215 Rice 0.104 Orange 0.135 Cereal 0.107 Fruit 0.251 Sugar 0.031 Avg. 0.235 Avg. 0.087 Processed meats Canned fruits Hot dogs 0.446 Peaches 0.417 Hamburger 0.578 Pineapple 0.402 Avg. 0.512 Applesauce 0.320 Avg. 0.380 Fresh meats Beef 0.120 Chicken 0.191 Liver 0.150 Avg. 0.154

10-3 P.37

The data on lead in foods are not comprehensive Kello and Kostial30 have shown that milk in­ enough to permit construction of a spectrum of creases lead absorption in 6-week-old rats. Fasting levels ranging from fresh to canned and charac­ enhances lead absorption in mice.20 Low dietary terization of the resulting lead exposure. It must levels of calcium, iron, zinc, copper, selenium, and suffice to state at this point that some fraction of die­ vitamin D have been reported to enhance lead tary lead probably is indirectly of airborne origin. absorption.31-32 It has also been demonstrated that rats on an iron-deficient diet accumulate more lead 10.2.2.2 THE RELATIONSHIP OF ORAL IN­ in their bodies than do rats on an iron-sufficient TAKE TO BLOOD LEAD LEVELS diet.33 Table 10-2 presents the data of Barltrop34 It has been demonstrated repeatedly that blood relating to the effects of various nutritional factors lead levels increase when the oral intake of lead in­ on lead absorption as reflected in blood lead levels. creases, but a quantitative expression of this It should be noted that these studies are short term relationship has not been determined. Studies from studies obtained over a period of 48 hr. various parts of the world, as noted below, have shown that the increase in the blood lead for each TABLE 10-2. EFFECT OF DIFFERENT DIETS ON LEAD 100 fig of lead ingested daily ranges from less than 6 ABSORPTION EXPRESSED AS THE RATIO OF MEAN RETENTION FOR EXPERIMENTAL AND CONTROL to more than 18 /ug/dl. SUBJECTS34 It is important to point out that the high end of this range was gathered using subject groups whose die­ Ratio ol mean retention of lead tary intake may be of questionable relevance to the (experimental control) Diet Blood Kidneys Femur Liver general population. Tepper and Levin25 employed adult females in their study while Coulston et al.26 Low protein . 5.1 2.5 2.8 2.2 High protein 1 3.7 2.6 1 used an adult prison population. These findings are Low fat 1 1 1 1 not only in contrast to those from earlier U.S. studies High fat 9.6 7.6 4.8 4.2 but also to the results of a number of European Low minerals 17.7 11.9 13.7 8.8 studies based on the general population.27 The Euro­ High minerals 0.2 0.2 0.1 0.1 pean data are more consistent with a contribution of Low fiber 1 1 1 1 1 1 1 1 about 6 figld\ to the blood lead level per 100 fig High fiber Low vitamins 1 1 1 1 daily oral intake of lead; a similar level is reported High vitamins 1 1 1 1 by Kehoe.57 Children, particularly infants, absorb a larger percentage of lead than do adults. Consequently, the The absorption of lead in paint chips has received contribution of dietary lead to blood lead levels attention because ofthe risk for young children who probably is less for adults, but definitive data are not tend to ingest this material. Recent data from rat available. studies indicate that lead chromate and lead naphthenate incorporated into dried paint films are 10.2.2.3 ANIMAL STUDIES substantially available for absorption, although the The absorption of lead from food and changes in absorption rate is 30 to 50 percent what it is for lead absorption with age have been investigated in many naphthenate in oil or for lead nitrate in aqueous 35 36 animal studies; the usual values found ranged be­ solution. - The absorption of lead as a function of 37 tween 5 and 10 percent. However, Kostial et al.28 chemical form is shown in Table 10-3. demonstrated that 5- to 7-day-old rats absorb at 203 TABLE 10-3. PERCENTAGE ABSORPTION OF DIFFERENT least 55 percent of single oral tracer doses of Pb, LEAD COMPOUNDS RELATIVE TO LEAD ACETATE37 and Forbes and Reina29 reported that in rats the 2l2 gastrointestinal absorption of tracer doses of Pb, Lead compound Absorption. % 85Sr, and 59Fe was high prior to weaning but Control (no lead) 4 decreased rapidly thereafter. The absorption rate for Metallic lead (180 to 250/xm) 14 lead was 83 percent at 16 days; it then decreased Lead chromate 44 gradually to 74 percent on the day of weaning (22 Lead octoate 62 days) and rapidly thereafter to about 16 percent at Lead naphthenate 64 67 89 days. Although there may be some question about Lead sulfide Lead thallate 121 the applicability of these data, they are consistent Lead carbonate (basic) 164 with results reported from studies of young children.

10-4 10.2.3 Cutaneous Absorption ever, whenever short-term high levels oflead intake Absorption through the skin is of importance only are superimposed on such a long-term ingestion pat­ in the case of organic compounds of lead, particu­ tern. larly the lead alkyls and lead naphthenates.38-39 Soon after tetraethyl lead was introduced into com­ 10.3.1 Human Studies mercial use, Eldridge39 reported that it was ab­ Autopsy data have shown that lead becomes sorbed through the skin with great facility in both localized and accumulates in bone. This accumula­ dogs and guinea pigs. The presence of gasoline has tion begins in fetal life,44-45 since lead is readily been said to delay the penetration of tetraethyl lead transferred across the placenta. The concentration through the skin,40 although it has no effect on its of lead in the blood of newborn children is similar to uptake by the lungs.41 In rats, five cutaneous or sub­ that of their mothers,46-47 and the distribution of cutaneous applications on alternate days of lead ace­ lead in fetal tissue is similar to that of adults.45 tate or lead naphthenate produced, compared with The total content of lead in the body may exceed unexposed animals, a decrease in ALAD in liver, a 200 mg in men aged 60 to 70 years, but in women it decrease in liver and body weight, and distribution is somewhat lower. Calculations by several in­ of lead in assayed body tissues. Lead content was vestigators48 show that in nonoccupational^ ex­ highest in kidney. Lead naphthenate was considered posed adults 94 to 95 percent of the total body by the investigators to be more toxic than lead ace­ burden is in the bones.44-49-50 These reports not only tate because of more pronounced skin reactions, reaffirm the affinity of bone for lead, but also pro­ higher lead accumulation in brain, and the occur­ vide evidence that the concentrations of lead in rence of a paralytic syndrome before death in two bones increase at least until middle age (50 to 60 42 animals. years old).48-51 On the contrary, neither soft tissues The rate of absorption of tetraethyl lead and in­ nor blood show age-related changes in lead con­ 52 53 organic compounds through the skin was studied by centration after age 20. - Thus, it seems that the Lang and Kunze.43 They applied solutions of lead skeleton is a repository that reflects the long-term acetate, lead orthoarsenate, lead oleate, and accumulative exposure to lead, whereas body fluids tetraethyl lead to the bare skin of a number of rats and soft tissues equilibrate rather rapidly and reflect and measured the amount oflead in the kidney as an only recent exposures. index of absorption. In all cases, the amount of lead The concentration of lead in the blood is utilized in the kidney was greater than in controls; tetraethyl as an index of exposure to assess conditions con­ lead produced the greatest difference. Ifthe skin was sidered to represent a risk to health.54 Plasma lead traumatized before the lead solutions were applied, concentrations have been shown to be constant at 2 there was a threefold or fourfold increase in renal to 3 /ug/dl over a range of 10 to 150 /ug/dl whole lead concentration. It is likely that absorption blood.55 Recent studies have indicated that lead is through unabraded skin by various lead compounds bound primarily to erythrocyte protein, chiefly is primarily dependent on their relative lipid hemoglobin, rather than to stroma.56 solubilities. Rabinowitz used a stable lead isotope tracer Because of the difficulty, particularly in tetra- (204Pb) to determine the rate of equilibration of ethyl-lead-contaminated atmospheres, in attempting blood lead with input.21 He found that in human to separate cutaneous exposure from respiratory ex­ subjects with a constant daily oral intake of 204Pb, a posure, the role of cutaneous lead absorption in rela­ virtually constant concentration was measured in tion to blood lead levels is still unclear. blood after about 110 days. When lead was removed from the diet, the concentration in the blood disap­ 10.3 DISTRIBUTION peared with a half time of approximately 19 days. When a single dose of lead enters the body, it is Tola et al.57 reported that the concentration of lead distributed initially in accordance with the rate of in the blood rises fairly rapidly to a new steady-state delivery of blood to the various organs and systems. level in about 60 days when men are introduced into The material is then redistributed to organs and an occupational lead-exposure situation, a situation systems in proportion to their respective affinities similar to that cited for exposure chambers in clini­ for lead. When daily ingestion is consistent for an cal studies.58 extended period, a nearly steady state is achieved Although the body burden of lead increases with respect to intercompartmental distribution. throughout life,48-50-52 measurements of specific The steady-state condition will be disturbed, how­ organs and systems show that the total burden is

10-5 P.39

divided between two general pools within the body. much more gradual response, with a delay of The major portion of the lead is contained in bone. approximately 35 days. This pool is clearly highly accumulative and, as a consequence, lead accumulates here rather con­ 10.3.2 Animal Studies sistently and continuously. The second pool com­ Administration of a single dose of lead to rats pro­ prises other organs and systems and accumulates duces high initial concentrations of lead in soft much less. The levels of lead in this pool tend to tissues which then fall rapidly as the result of excre­ stabilize early in adult life and thereafter demon­ tion and transfer to bone.66 The distribution charac­ strate a turnover rate sufficient to prevent accumula­ teristics of lead within the animals' bodies were tion. found to be independent of the dose over a wide Since the organs and systems that contain the range. Castellino and Aloj67 described the rate con­ relatively mobile lead pool are of greater toxicologi­ stants for the elimination of lead from various cal significance, it is clear that a mobilizable or tissues in rats following a single dose. Lead was exchangeable lead burden is a more important con­ eliminated from bone much more slowly than from cept than is total body burden. In this connection, other tissues. Bolanowska et al.68 reported that the chelatable urinary lead has been shown to provide rate of elimination of a single dose of lead from rats an index of the mobile portion of the total lead became slower with time, reflecting progressively burden.59-60 Among adults in the general population decreasing mobility ofthe residual body burden. there are no age-related differences in con­ Goldstein et al.69 sacrificed 21-day-old rats 24 centrations of lead in whole blood or in blood hr after intravenous injection of various single serum. Thus, in a general way, the blood lead level is doses (1. 50, 200 fig) of labeled lead (210Pb) and an indicator of the concentration of lead in soft found that the concentration of radioactivity in the tissues, and the changes in blood lead levels ob­ brain was directly proportional to the blood radio­ served when there are changes in exposure levels activity. Studies of OTuama et al.70 indicate, probably reflect similar changes in some organs and however, that this process may not be one of simple soft tissues. passive diffusion of lead into neural tissue. These latter investigators sacrificed guinea pigs at 5, 60, Lead exposure causes the development of nuclear and 240 min. after the intravenous injection of tracer inclusion bodies containing lead in both man61"63 doses of lead (2l0Pb) in both subacutely intoxicated and animals. Although they seem to occur most fre­ (155 mg PbCOj/day for 5 days) and control animals. quently in the kidney, they have been found in other Radioactivity in barrier tissues (such as choroid organs as well. plexus and meninges) rose rapidly, with concentra­ The concentrations of lead in deciduous teeth are tions ranging to more than ten times the of interest because tooth analysis represents a nonin­ simultaneous brain radioactivity. Subsequently, vasive technique and because teeth provide a record there was a fall in the barrier tissue levels, but brain of long-term lead exposure. The dentine is par­ levels remained fairly constant and low throughout ticularly useful in this respect because it is laid down the period of the study. The apparent discrepancies from the time of eruption to the time the tooth is may be explained in large part by the differences in shed. Concentrations of lead in dentine are reported the ages of the animals as well as other factors, in­ to be considerably lower in suburban school cluding species differences, time of sacrifice, or a children than in children residing in areas of high more complex mechanism of distribution. 64 lead exposure. Rather striking age-related differences in the dis­ Primarily because of the relative ease with which tribution and retention of lead in rats have been ob­ 71 203 hair can be collected, there have been some studies served. Elimination of a single tracer dose of Pb of the possible use of hair lead as an index of ex­ from the whole body, blood, and kidney occurred posure. These studies have not been sufficient, more rapidly in adult than in suckling rats. In suck­ however, to provide significant information on the lings there was a slight increase with time in the 203 relationship between hair lead concentrations and Pb content of the brain following administration the amount of exposure. Rabinowitz et al.65 fed of the dose, whereas the content in other soft tissues labeled lead (204Pb) to 3 subjects daily for approxi­ decreased with time. mately 100 days. Levels of isotope in the blood were The intracellular distribution of lead has been immediately elevated but in facial hair there was a studied in rat tissue, mainly by cell-fractiona-

10-6 tion techniques.72-73 Membranes, especially Thus, gastrointestinal transit appears to play an im­ mitochondria, have shown an affinity for lead. Little portant role in the rate of excretion in feces of lead is found in lysosomes,73 however, in contrast systemically administered lead. with the intracellular distribution of many other The clearance of lead from the blood of man into metals, e.g., mercury, copper, and iron. urine was found by Vostal82 to be proportional to There are few studies of target organs in which the rate of creatinine excretion, with urinary lead lead concentrations at the site ofthe effect have been extrapolating to zero at zero creatinine. specifically determined. In particular, direct assess­ The characteristics of urinary lead excretion may ment of lead level in bone marrow is difficult to car­ be affected by the chemical form of lead. Whereas ry out, although the sensitivity of the hematopoietic all of the lead in urine of subjects with normal ex­ system to lead has been extensively investigated. posure can be precipitated by the addition of agents Formation of nuclear inclusion bodies is observed in such as oxalate, phosphate, or carbonate, only one- rats with renal lead concentrations of about 10 third to two-thirds of the lead in the urine of lead mg/kg (wet weight) of kidney.74 Other effects of lead workers is available for precipitation.83 These were found to occur only at higher levels of organ results suggest the presence ofa stable lead complex concentration. Death in cattle is associated with lead in the urine of exposed workers. levels of about 50 mg/kg (wet weight) of kidney cor­ Lead is excreted in sweat as well as urine. tex.75 Schiels84 reported that ingestion of lead acetate in­ The concept of estimating the lowest level of creased the lead concentration of sweat twofold to metal accumulation that results in adverse effects in fourfold. Schroeder and Nason85 found the con­ a target organ has not been well explored in the case centration of lead in the sweat of lead-intoxicated of lead. This is in contrast with cadmium, for which subjects to be similar to that in urine. estimates have been made of the minimum con­ Since studies of net lead retention suggest that, at centrations in the kidney cortex at which evidence of low level exposures, higher intakes are followed by renal damage appears.76 higher rates of excretions5"7-17-59 and that lead excre­ tion appears to be disproportionately low in cases of 10.4 ELIMINATION high-level exposure, there is not yet a predictable The major portion of excreted lead appears in relationship between increases in lead exposure and urine and feces, but lesser quantities are removed via in lead excretion. sweat, hair, nails, and exfoliated skin. 10.4.2 Animal Studies 10.4.1 Human Studies The relative importance of lead excretion from Fecal excretion represents the major route of blood into urine and feces varies with the species organic and inorganic lead elimination. The rate of tested. Within 12 hr, 7.4 percent of an intravenous fecal lead excretion has been reported to be 100 dose appeared in the feces of rats compared with a times the rate of elimination in urine;77-78 however, recovery of 2.3 percent trom urine.86 In sheep, also, most of the lead in feces represents metal that has fecal elimination is more rapid than urinary excre­ not been absorbed. tion of lead.87 In contrast, urinary excretion was re­ Rabinowitz et al.79 studied the excretion of tracer ported to be two times greater than fecal excretion in lead from the blood ofa nonoccupational^ exposed baboons.89 human subject. Urinary and fecal excretion of 204Pb There are indications that most ofthe translocated from the blood amounted to 38 and 8 fig/day, ac­ lead (from blood to intestine) is derived from bile. counting for 76 and 16 percent, respectively, ofthe Of the 7.5 percent of an intravenous dose of lead measured recovery. The crude estimation of lead in acetate excreted by sheep in the feces within 6 days, hair, nails, and sweat yielded a value of 4 fig (8 per­ 81 percent originated in bile.87 Similar results were cent). The urinary excretion was similar to the obtained from rats.86-89 Although species differences average daily lead excretion of 31 /ug/day reported in gastrointestinal transit time and the presence ofa by Teisinger and Srbova.80 Booker et al.81 ad­ gallbladder can explain differences in the rate of ap­ ministered lead(212Pb) intravenously to 2 human pearance ofa single injected dose of lead in the feces, subjects and then recovered 4.4 percent of the dose these factors do not account for differences in the in urine during the first 24 hr. Lead was not relative amounts of steady-state lead elimination in detected in feces. During the second 24 hr, about urine and feces. 1.5 percent was measured in both urine and feces. Measurements of lead clearance into the urine of

10-7 P.41

animals, like those in man, require an accurate organic lead to triethyl lead ranged from 67:1 to measurement of the free lead concentration in 18:1 in the urine. This ratio did not reflect the ratio blood. Since most of the lead in blood is bound to of inorganic to triethyl lead in tissues, including the red cells and to plasma proteins, this measurement is brain where the ratio was approximately 1:1. virtually impossible. Whether the renal tubule takes an active part in 10 6 METABOLIC CONSIDERATIONS IN lead excretion is open to question. More recently, it THE IDENTIFICATION OF SUSCEPTIBLE has been found that the renal tubule cell transports SUBGROUPS IN THE POPULATION lead into the urine,90 perhaps because of the pre­ sence of lead-binding ligands in the tubular cell. The discussion on the metabolism of lead has up These observations demonstrate that the renal ex­ to now only tangentially specified differences in cretion of lead involves more than filtration of the metabolism between children and adults (Section metal at the glomerulus. It is likely that the 10.2.2). There are, however, physiological dynamics responses of secretory and reabsorptive processes to of child growth and development that have signifi­ increased circulating lead levels contribute to the cant implications for the increased risk of children relative constancy of urinary excretion in humans5"7 exposed to lead. There are, as well, differences be­ and in laboratory animals74 that were exposed to tween children and adults in the intake, desposition, high doses of lead. etc., of lead. Metabolic, physical, and other differences be­ tween children and adults that must be considered 10 5 ALKYL LEAD METABOLISM include: (1) children have considerably less surface The toxic effects caused by tetraethyl lead and area than adults, e.g., a 2-year-old child has one- tetramethyl lead are not produced by the tetraalkyl third the surface area of an adult; this parameter is compounds themselves, but rather by the trialkyl not known to be directly related to the risks associ­ derivatives formed by dealkylation in the liver.86-91 ated with lead exposure;98 (2) there is greater lead Tetraethyl lead is converted primarily to triethyl intake by infants on a per-unit-body-weight basis, lead and partly to inorganic lead.92 Triethyl lead which is probably related to greater caloric and concentrates in organs and disappears very slowly. water requirements; (3) there is greater intake in Even after several days, there is no significant reduc­ children as well as net absorption (Section 10.2.2). tion. Tetramethyl lead is much less toxic than resulting from greater net respiratory intake along tetraethyl lead, probably because it is dealkylated to with greater net absorption and retention from the the trialkyl toxic form much more slowly than is gastrointestinal tract: (4) the rapid growth rate of tetraethyl lead.93 children may reduce the margin of safety against a variety of stresses, including iron deficiency, etc.; (5) Since both these compounds have toxic and 99101 biochemical effects unlike those of inorganic lead, dietary habits of children in some respects are the biochemical indices used in assessing inorganic quite different from those of adults; normal hand-to- lead exposure would not be expected to have the mouth activity such as thumb sucking occurs as well same significance in assessing exposure to organic as the habit of retrieving dirt-contaminated lead. Indeed, in cases of severe, acute tetraethyl lead foodstuffs: (6) in children the likelihood of protein, poisoning, urinary coproporphyrins and ALA excre­ calcium, and iron deficiency is so great relative to tion are not usually elevated, and free erythrocyte intake that a negative balance in these factors may exist; (7) in very young children metabolic path­ porphyrins are only moderately and inconsistently 99 01 elevated.94-95 These biochemical tests are therefore ways ' are known to be incompletely developed, of little use in short-term exposure situations. In e.g., the blood-brain barrier in newborns; and (8) partitioning of lead in the bones of children is long-term exposure situations, however, it is possi­ I00 01 ble that some of them may be useful. Indeed, Robin­ different from that of adults. ' Only 60 to 65 son96 has shown that in industrial workers exposed percent of the lead body burden is in the bones of to tetraethyl lead, urinary excretion of ALA is in­ children. More important is the possible lability of creased, but not to the same degree as in workers ex­ the bone fraction of lead in children, particularly in the case of coexisting calcium deficiency. Rosen and posed to inorganic lead who have similar levels of 02 total urinary lead excretion (organic plus inorganic). Wexler' find an increasing resorption oflead in rat Bolanowska et al.97 demonstrated that in three fatal bone organ culture when calcium in the medium is cases of tetraethyl lead poisoning the ratio of in­ reduced.

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The effects of chelating agents on the Commission ofthe European Communities, p. 591-603. tissue distribution and excretion of lead. Toxicol. Phar­ 48. Barry, P. S. and D. B. Mossman. Lead concentrations in macol. /8:296-310, 1971. human tissues. Brit. J. Ind. Med. 27:339-351, 1970. 67. Castellino, N. and S. Aloj. Kinetics ofthe distribution and 49. Horiguchi, S. and T. Utsunomiya. An estimate ofthe body excretion of lead in the rat. Brit. J. Ind. Med. 2/:308-314, burden of lead in the healthy Japanese population. An at­ 1964. tempt to assume absorption and excretion of lead in the 68. Bolanowska, W., J. Piotrowski, and B. Trojanowska. The healthy Japanese population, Part 2. Osaka City Med. J. kinetics of distribution and excretion of lead (Pb210) in 79:1-5, 1973. rats. In: Proceedings of the 14th International Congress of 50. Schroeder, H. A. and I. H. Tipton. The human body Occupational Health, Madrid, September 16-21, 1964. p. burden of lead. Arch. Environ. Hlth. 77:965-978, 1968. 420-422. 51. Gross, S. B., E. A. Pfitzer, D. W. Yeager, and R. A. Kehoe. 69. Goldstein, G. W., A. K. Asbury, and I. Diamond. Lead in human tissue. Toxicol. Appl. Pharmacol. Pathogenesis of lead encephalopathy. Arch. Neurol. 32:638-651, 1975. 37:382-389, 1974.

10-10 70. O'Tuama, L. A., C. S. Kim, J. Gatzy, M. R. Krigman, and 85. Schroeder, H. A. and A. P. Nason. Trace element analysis P. Mushak. The distribution of inorganic lead in guinea in clinical chemistry. Clin. Chem. 7 7:461 -473, 1971. pig brain and in neural barrier tissues in cortical and lead- 86. Costellino, N., P. Lamanna, and B. Grieco. Biliary excre­ poisoned animals. Tox. Appl. Pharm. 36:1-9, 1976. tion of lead in the rat. Brit. J. Ind. Med. 23:237-239, 1966. 71. Momcilovic, B. and K. Kostial. Kinetics of lead retention 87. Blaxter, K. L. and A. T. Cowic. Excretion of lead in the and distribution in suckling and adult rats. Environ. Res. bile. Nature. 757:588, 1946. 8:214-220, 1974. 88. Cohen, N., M. Eisenbud, and M. E. Wrenn. The Retention 72. Costellino, N. and S. Aloj. Intracellular distribution of and Distribution of Lead-210 in the Adult Baboon. An­ lead in the liver and kidney ofthe rat. Brit. J. Ind. Med. nual Rept. to the U.S. Atomic Energy Commission (Oct. I, 26:139-143, 1969. 1962 - Sept. 30, 1967). U.S. Dept. of Interior. Cincinnati, 73. Barltrop, D., A. J. Barret, and J. T. Dingle. Sub-cellular Ohio 1967. 218 p. distribution of lead in the rat. J. Lab. Clin. Med. 89. Cikrt, N. Biliary excretion of 2o*Hg, MCu, 52Mn, 2iopb in 77:705-712,1971. the rat. Brit. J. Ind. Med. 29:74-80, 1972. 74. Goyer, R. A., D. L. Leonard, J. F. Moore, B. Rhyne, and 90. Vostal, J. Mechanisms of renal lead excretion. Biochem. M. R. Krigman. Lead dosage and the role of the in­ Pharmacol. Conf. Issue. 2:207, 1963. tranuclear inclusion body. Arch. Environ. Hlth. 91'. Cremer, J. E. and S. Callaway. Further studies on the tox­ 20:705-711, 1970. icity of some tetra- and trialkyl lead compounds. Brit. J. 75. Allcroft, R. and K. L. Blaxter. Lead as a nutritional Ind. Med. /8:277-282, 1961. hazard to farm livestock. V. The toxicity of lead to cattle 92. Bolonowska, W. Distribution and excretion of triethyl and sheep and an evaluation ofthe lead hazard under farm lead in rats. Brit. J. Ind. Med. 25:203-208, 1968. conditions. J. Comp. Path. Ther. 60:209-218, 1950. 93. Cremer, J. E. Toxicology and biochemistry of alkyl lead 76. Friberg, L., M. Piscator, G. Nordberg, and T. Kjellstrom. compounds. Occup. Health Rev. 77:14-19, 1965. Cadmium in the Environment, 2nd Ed. Ohio, Chemical 94. Gutniak, O., H. Koziolowa, and E. Kowaiski. Free pro­ Rubber Co. 1973. toporphyrin content of erythrocytes in chronic tetraethyl 77. Kehoe, R. A. Normal metabolism of lead. Arch Environ. lead poisoning. Lancet. 7:1 I 37-1 I 38, 1964. Hlth. 8:232-235, 1964. 95. Beattie, A. D., M. R. Moore, and A. Goldberg. Tetraethyl- 78. Kehoe, R. A. Metabolism of lead under abnormal condi­ lead poisoning. Lancet. 2:12-15, 1972. tions. Arch. Environ. Hlth. 8:235-243, 1964. 96. Robinson, T. R. Delta-amino levulinic acid and lead in 79. Rabinowitz, M. B., G. W. Wetherill, and J. D. Copple. urine of lead antiknock workers. Arch. Environ. Hlth. Lead metabolism in the normal human: Stable isotope 28:133-139, 1974. studies. Science. 782:725-727, 1973. 97. Bolanowska, W., J. Piotrowski, and H. Carczynski. 80. Teisinger, J. and J. Srbova. The value of mobilization of Triethyllead in the biological material in cases of acute lead by calcium ethylene-diamine-tetraacetate in the diag­ tetraethyllead poisoning. Arch. Toxicol. 22:278-282, nosis of lead poisoning. Brit. J. Ind. Med. 76:148-152, 1967. 1959. 98. Ziegler, E. E., B. B. Edwards, R. L. Jensen, K. P. Booker, D. V., A. C. Chamberlain, D. Newton, and A. N. Mahaffey-Six, and J, J. Fomon. Absorption and retention B. Stott. Uptake of radioactive lead following inhalation oflead by infants. Ped. Res. 12. In Press. 1978. and injection. Brit. J. Radiol. 42:457-466, 1969. 99. National Research Council. Recommendations for the prevention of lead poisoning in children. National 82. Vostal, J. Study of the renal excretory mechanisms of Academy of Sciences. Washington, D.C. July 1976. heavy metals. 15th International Congress of Occupa­ 100. Lin-Fu, J. S. Vulnerability of children to lead exposure tional Health, Vienna, 19-24 September, V. 3. 1966. p. and toxicity. N. Engl. J. Med. I. 289(23): 1229-1233, 1973. 61-64. 101. Lin-Fu. J. S. Vulnerability of children to lead exposure 83. Dinischiotu, G. T., B. Nestorescu, J. C. Radielescu, C. and toxicity. N. Engl. J. Med. II. 289(24):1289-1293, Jonescu, N. Preda, and G. Hutza. Studies on the chemical 1973. forms of urinary lead. Brit. J. Ind. Med. / 7:141-145. I960. 102. Rosen, J. F. and E. E. Wexler. Studies of lead transport in 84. Schiels, D. O. Elimination of lead in sweat. Aust. Ann. bone organ culture. Biochem. Pharmacol. 26:650-652, Med. 3:225, 1954. 1977.

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11. BIOLOGICAL EFFECTS OF LEAD EXPOSURE

11.1 INTRODUCTION cumulative or additive adverse effects of minimal As noted in Chapter 2, air-quality criteria docu­ biological response in a number of systems. ments present the scientific knowledge of the rela­ 11.2 CELLULAR AND SUBCELLULAR tionship between pollutant concentrations and their EFFECTS OF LEAD adverse effects on public health and the environ­ 11.2.1 Effects on Enzymes ment. This chapter addresses the important human health and biological effects of lead exposure. In general, the effects of lead on enzymes may be Section 11.2 treats the biochemical and pathologi­ manifested in several ways. Lead, in common with a cal basis ofthe various health effects of lead, center­ number of other metals, has an affinity for a number ing on enzymology and the subcellular and cellular of complexing groups resident in the structure of aspects of lead effects on the various organ systems. many biomolecular entities, such as imidazole Section 11.3 presents a brief overview of clinical nitrogen, the cysteine sulfhydryl group, and the e- lead poisoning — that is, lead exposure leading to a amino group of lysine. An effect may be imparted, constellation of adverse health effects that require therefore, by binding-site competition with the na­ medical intervention. It is not the purpose of that tive ion, by perturbation of the structural integrity of subsection to suggest that airborne lead invariably enzymes, or by the impediment of substrate-enzyme induces clinical lead poisoning as defined in this binding. document; rather, it seeks to state the consequences Cellular damage caused by lead may also permit to health, both immediate and long term, ofthe up­ the movement of enzymes into the circulatory per range of exposure to this pollutant regardless of system, with a resulting elevation of enzyme activity its source and to state this in a discrete portion ofthe in, for instance, plasma. document. The effects of lead on enzymes and enzyme The respective sections on organ systems have systems have been studied in both animals and ex­ been ordered according to the degree of known posed human subjects and in vitro and in vivo. vulnerability to lead of each system. The emphasis is Clearly, many ofthese studies in the literature are of not only on the three systems classically considered marginal relevance to this particular document and most sensitive — hematopoietic, nervous, and are briefly summarized for reference reading with­ renal — but also on reproduction and development out evaluation. in view of lead's effects on the fetus, and, therefore, On the other hand, a number of other enzyme on pregnant women. Some effects can be considered systems are of such distinct relevance that they are to involve a number of organ systems, and the better elaborated in the specific sections on organ available data on multisystemic effects are presented effects. For example, the enzymology relating to the in the final subsection. heme biosynthetic pathway is discussed in Section Subdividing the chapter on the health effects of 11.4. lead into organ systems was done for the purpose of Enzymes that have been shown to be affected by easier discussion. It must be kept in mind that, in lead in animal studies are presented in Table 11-1, reality, all systems function in delicate concert to and results of studies on enzymes in humans are pre­ preserve the physiological integrity of the whole sented in Table 11-2. organism. Furthermore, all systems are interdepen­ dent in the organism, so that not only are effects in a 11.2.2 Organellar and Cellular Effects critical organ transmitted to other systems but also It is of interest to duscuss briefly the subcellular low-level effects, which may be construed as less im­ distribution of lead before further comment is made portant in a single specific system, contribute to the on cellular effects.

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TABLE 11-1. ENZYMES AFFECTED BY LEAD IN ANIMAL Similarly, Barltrop et al.32 measured 203Pb in STUDIES heart, liver, kidney, and spleen following in­ traperitoneal (i.p.) administration. Their results in­ Enzyme Effect on activity Reference dicated that most of the lead accumulated in the 1 Lipoamide dehydrogenase Inhibited mitochondria. DNAase Enhanced 2.3 A detailed study ofthe rat kidney by Goyer et al.33 Serum glutamic oxaloacetic Enhanced and 4-7 showed that over a protracted period the cell transaminase (SGOT) transitory nucleus accumulated the highest proportion of lead.

Serum glutamic pyruvic Enhanced and 4-7 Under lead challenge, a cellular reaction typical transaminase (SGPT) transitory of a variety of animal species is the formation of in­ tranuclear inclusion bodies, the early experimental 7 Serum alkaline phosphatase Lowered history of which has been reviewed by Goyer and (AP) i Moore.34 The presence of considerable lead in these Erythrocyte and liver AP Variable 7.8 bodies has been verified by X-ray microanalyses;35 Acid phosphatase Quenched or 7,9,10 ultrastructural studies show that this entity consists markedly inhibited of a rather dense core encapsulated by a fibrillary envelope. Catalase Variable 11-13 The work of Goyer,33 36 indicates that these inclu­ Chollnesterase Markedly inhibited 5.6.14 sion bodies are a complex of lead and protein, the a-Mannosidase Increased 15 protein moiety having characteristics of the residual /3-Acetyl glucosaminadase Increased 15 acidic fractions of proteins in normal nuclei. The morphological integrity of these inclusion bodies Succinate oxidase Decreased 16 collapses on treatment in vitro with metal chelants Cytochrome c reductase Decreased 16 such as EDTA. A role for the inclusion body as a Glutamate dehydrogenase Decreased 16 cellular protective mechanism during transcellular lead transport has been postulated.36 Cytochrome oxidase Decreased 16 How the localization of lead in nuclear inclusions Rat brain adenyl cyclase Decreased 17 relates to nuclear function has not been established; 37 /3-Glucuronidase Elevated 18 however Choie and Richter have shown that i.p.- administered lead enhances DNA synthesis and /3-Galactosidase Elevated 18 proliferation of renal tubular cells. The effects of lead on cell division are detailed in Section 11.2.4. TABLE 11-2. ENZYMES AFFECTED BY LEAD IN HUMAN STUDIES Disaggregation occurs in the ribosome in the pre­ sence oflead.'38 Enzyme Effect on activity Reference Animal experiments and human studies, mainly SGOT and SGPT Enhanced 19-21 centered on cellular energetics and morphological SGOT and SGPT No effect 22 aberrations, have shown mitochondria to be highly Serum alkaline phosphatase Reduced 23 sensitive to lead. Teras and Kakhn39 showed de­ Acid phosphatase No effect 24.25 creased respiratory rates in mitochondria of rabbit Aldolase Enhanced 26 Cholinesterase Inhibited 27-29 tissue under chronic lead challenge using a- Glutathione reductase Enhanced 30 ketoglutarate, succinate, and pyruvate as substrates. Glucose-6-phosphate Inhibited 30 Phosphorylation was also retarded. dehydrogenase A marked sensitivity ofthe pyruvate-NAD reduc­ tase system in kidney mitochondria of lead-intoxi­ Castellino and Aloj,31 using 210Pb, found a cated rats is suggested by the work of Goyer and 40 decrease in radioactivity over a 24- to 72-hr time in­ Krall, who note impairment of pyruvate-depen- terval in the nuclear fraction and an increase of dent respiration using ADP/O ratios and respiratory 210Pb in the mitochondria fraction using liver. Over control rates (RCR's) as indices. Succinate-medi- the same time interval, an increase in radioactivity ated respiration in lead-intoxicated rats, however, occurred in the kidney in both nuclear and was not different from that of control animals. mitochondrial fractions, and there was a decrease in Rhyne and Goyer41 state that their observations of microsomes. Mitochondrial binding was particu­ decreased oxygen uptake rates for both State III and larly strong. IV in succinate-dependent respiration in

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mitochondria of the kidney from lead-intoxicated erythrocyte) protoporphyrin (FEP) and urinary animals may be evidence of decreased succino-ox- coproporphyrin. The discussion of the idase enzyme, which would also be consistent with hematopoietic system in Section 11.4 includes treat­ decreased mitochondrial protein. ment of this relationship. Walton42 reported an accumulation of lead in gran­ In addition, the intramitochondrial stages of heme ules produced in isolated rat-liver mitochondria synthesis have been suggested to have an intermedi­ following incubation in media containing lead, with ary role in introcellular metabolism and are proba­ the lower end of the range of the free lead level bly required for the continued transfer of iron from employed approaching that found in lead-poisoning extracellular sites to normoblasts of victims. It was noted that lead-rich granules, unlike reticulocytes.4546 those obtained with calcium, were not dispersed The in vivo effects of lead on erythrocytes in after treatment with dinitrophenol. This finding humans include: accumulation of lead, increased would indicate that lead removal after deposition is osmotic resistance, increased mechanical fragility, difficult. The above observations and other studies increased glucose consumption, increased potassium prompt the suggestion that lead effects are twofold: loss in incubation, decreased sodium- and (1) energy diversion to the active accumulation of potassium-dependent ATPase activity in membrane lead would prevent ATP synthesis and the preserva­ fragments, and elevation in the number of immature tion of ionic gradients in the membrane; and (2) the red cells.4749 A more detailed discussion of chemical action of lead would promote ATP hy­ erythrocyte-lead relationships is given in the drolysis, and lead would complex with essential SH hematopoietic section. groups of mitochondrial enzymes and interact with Jandl and coworkers50 demonstrated that the up­ anions. The net result is the inability of cells to take of 59Fe by human reticulocytes was almost com­ maintain themselves structurally and metabolically. pletely inhibited by 5 x IO4 M lead and that its in­ Recent studies by Kimmel et al.43 on the chronic corporation into hemoglobin was almost entirely long-term exposure of rats to lead showed that sub­ prevented. This resulted in an elevated level of iron cellular effects of lead on the renal system were ap­ in the erythrocyte membrane. parent when comparatively low levels of lead (5, 50, The major cellular pathology of concern in the and 250 ppm in drinking water) were administered kidney with reference to lead is that ofthe proximal prenatally and up to 9 months of age postnatally. convoluted tubular cells. Initial atrophy of the Light microscopy showed karyomegaly and epithelial cells in this region is followed by cell cytomegaly at all dose levels. Electron microscopic regeneration along with an increase in intertubular examination indicated swollen mitochondria, connective tissue, basement membrane thickening, numerous dense lysosomes, and intranuclear inclu­ and round cell proliferation. Tubular cell sion bodies at 50 and 250 ppm. Furthermore, con­ mitochondria swell and degenerate, as noted before, siderable alteration in the activity of heme bio­ and glomeruli show increased cellularity.51 Tubule synthetic pathway enzymes (8-ALA synthetase and cells also show the presence of nuclear inclusion ferrochelatase) was observed at 50 and 250 ppm. bodies, a description of which has been made (vide The blood lead values for the different dose regi­ supra). mens ranged from 5 figldl for control animals and In the suckling-rat model for lead encephalopathy 10 figldl for the 5 ppm group to 25 /ig/dl for the 50 employed by Pentschew and Garro5253 in which lead ppm group and 70 figldl for the 250 ppm animals. exposure of the pups is via milk from mothers fed Cramer et al.44 studied renal biopsy tissue of five lead carbonate, epithelial cell dysfunction in brain workers having varying periods of exposure to lead. capillaries is evidenced by abnormal permeability to Although the typical lead-induced nuclear inclusion Trypan Blue and Thorotrast (colloidal thorium bodies were found only in those with short exposure, dioxide). The lesion possibly centers on interference all subjects showed mitochondrial changes. with an energy-regulating mechanism peculiar to its Mitochondria in the tubular lining cells showed barrier function. swelling and distortion of cristae, with some of the Schlaepfer54 has suggested that the neuropathy of mitochondria transected by cristae. lead poisoning may be caused by initial damage to An important comment here relates to the indica­ the supporting cells of the nervous system. Dorsal tions of impaired mitochondrial function of root ganglion capsular cells show a proliferation and erythroid tissue in humans that can be assessed by accumulation of dense bodies in their cytoplasm that changes in levels of free erythrocyte (actually zinc microscopically possess the features of a heavy

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metal. It is possible that the metabolism ofthe cap­ cant increase in chromosomal damage in male sular cells is impaired, which then causes the workers exposed to lead oxide fumes in a shopbreak­ degeneration of associated neurons and axons and ing yard. These shipbreakers had blood lead values has a deleterious effect on the ganglion cells. A com­ ranging from 40 to over 120 figldl. Schmid et al.64 mon site for intoxication in both the capsular cells found no evidence of increased chromosomal aber­ and the Schwann cells of the peripheral nervous rations in peripheral lymphocytes, studied both in system has been suggested to account for both ax- vivo and in vitro, in lead manufacturing workers. 65 onal degeneration and segmental demyelination55 Furthermore, Bauchinger et al. found no abnor­ because these two cells have a common embryologi- malities in the chromosomes of policemen with ele­ cal origin.56 vated blood lead levels (20 to 30 percent above the Moore et al.,57 in a study ofthe cardiac effects of mean for the control group). lead in the drinking water of rats, found that when An increase in chromosomal aberrations in people rats were exposed to lead in drinking water at a level occupationally exposed to lead whose mean blood similar to levels previously found in Glasgow, Scot­ lead values were 38 to 75 figldl has been reported, land, there was a significant inhibition of cardiac however, by Forni and Secchi58 and by Schwanitz ferrochelatase and 8-aminolevulinic acid dehy­ et al.56 Moreover, Deknudt et al.60 reported dratase that was maximal after 6 months. Moreover, chromosomal damage in a group of 14 male workers electron microscopy revealed marked changes in with signs of lead poisoning. Although the workers myocardium and myocardial mitochondria. were exposed to zinc and cadmium as well as lead, the authors concluded that lead should be con­ sidered responsible for the aberrations. The study by 11.2.3 Effects of Lead on Chromosomes Forni and Secchi58 showed that the rates of The examination of chromosomes for damage is chromatid changes were higher in 65 workers with technically difficult. The evaluation ofthe relevance preclinical and clinical signs of lead poisoning but of many studies can therefore be equally difficult. were not significantly raised for workers with past Because the appropriate separation of chromosomes poisoning. Forni et al.62 also examined 11 subjects into two chromatids and equal redistribution of before and during initial occupational exposure to chromatids during cell division are necessary for the lead. The increase in the rate of abnormal chromatid reproduction of stable new cells for the maintenance metaphases (the separation ofthe pair of chromatids of healthy tissue, the implications of injury to during normal cell division) was doubled after 1 chromosomal material are profound, and interrup­ month of exposure, was further increased after 2 tion of the processes involved can be serious. Incor­ months, remained in this stage up to 7 months, and rect division of cells by the breakage of the then decreased. The fact that most alterations were chromatid, the migration of an inappropriate set of of the chromatid type, that is, occurring in cell chromatids into either portion of a dividing cell, the culture after DNA synthesis, indicates that these abnormal reproduction of the complementary new could be culture-produced aberrations and may not chromatid to complete a viable chromosome in the reflect a realistic in vivo situation. Also, a number of new cell, and other deviations from the normal pro­ participants dropped out in the later stages of this cess can produce abnormal cells. Such chromosomal study. Thus, the actual biological significance of aberrations can, therefore, be responsible for the these results is unknown. production of such serious consequences as genetic 66 defects in offspring ofthe affected organism. In a recent report, Bauchinger et al. found that chromosomal aberrations were significantly in­ In the last few years, a number of reports have creased in a group of 24 male workers occupied in been published on the chromosomal effects of ex­ zinc electrolysis and exposed to zinc, lead, and cad­ 3 cessive exposure to lead in animals and mium. The workers had clearly elevated blood lead 5668 humans. Although some of these reports have and blood cadmium levels in comparison with a con­ 6365 been essentially negative, others have concluded trol group. The authors emphasized the possibility that there is a definite increase in the number of of a synergistic effect of several metals on the chromatid and chromosome changes in subjects who chromosomes. They also pointed out the similarity 5662 are occupationally exposed to lead. Thus, the between this group and the group studied by Dek­ literature is controversial in regard to chromosomal nudt et al.60 in regard to exposure to a combination abnormalities induced by exposure to lead. of lead, zinc, and cadmium. Referring to studies in­ O'Riordan and Evans63 did not find any signifi­ dicating the mutagenicity of cadmium, Bauchinger

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and his colleagues66 were inclined to consider cad­ ing 1 percent lead acetate for more than 1 year mium as being mainly responsible for aberrations; developed renal adenomas or carcinomas with an in­ but Deknudt et al.60 concluded that the abnor­ crease in both size and incidence of carcinomas re­ malities found were caused mainly by lead rather lated to duration of exposure. than by combinations of the three metals. Subcutaneous or intraperitoneal injections of lead The question of whether chromosomal abnor­ phosphate repeated over a period of several months malities occur in humans as a result of lead ex­ also induced renal tumors. The total doses ad­ posure, either alone or in combination with other ministered varied between 120 and 680 mg lead in pollutants, remains unanswered.67 Furthermore, the the animals developing the tumors.7178 human health significance of chromosomal abnor­ In addition to renal neoplasms, tumors of the malities seen in lymphocyte cultures, a method used testes, the adrenal, thyroid, pituitary, and prostate in some of the studies reported, is not yet known.68 glands, and the brain have been reported in Wistar An assessment of the possible mutagenic effects of rats fed lead acetate.79 lead is further hampered by the technical difficulties The morphologic and co-carcinogenic effects of that are inherent in the study of chromosomes. lead on the respiratory system were studied by Kobayashi and Okamoto.80 Male and female golden 11.2.4 Carcinogenesis hamsters were given a combination of 1 mg lead ox­ Lead salts have been shown to be at least co-car­ ide and 1 mg benzo[a]pyrene intratracheally once cinogenic in rats and mice.69 The ultrastructure of weekly for 10 weeks; lung adenomas occurred in 11 experimentally lead-induced renal tumors in of the 26 animals within 60 weeks. One adenocar­ animals70 is characterized by cellular and nuclear cinoma of the lung was also observed. Any hypertrophy, the presence of numerous lysosomes differences in frequency of occurrence between and microbodies, and the absence ofthe infolding of males and females were not mentioned. Such tumors basal plasma membranes that is normally seen in did not occur in animals given the same dose of lead renal tubular lining cells. These tumor cells do not oxide or benzo[a]pyrene alone. It should be noted, contain intranuclear inclusion bodies, and the lead however, that because lead compounds are only a content of the tumors is less than that in adjacent small fraction of total particulates in air, there may renal cortex. Renal adenomas and carcinomas were be enough particulates even without lead for first observed in rats by Zollinger71 in 1953 follow­ benzo[a]pyrene to be adsorbed so as to cause in­ ing long-term injections of lead phosphate. Later creased carcinogenicity. 72 Kilham et al. reported similar tumors in wild rats Tetraethyl lead (TEL) [Pb(C2H5)4] is an impor­ believed to have been exposed to lead fumes from tant, widely used, antiknock additive for motor burning refuse in a city dump. Lead-induced renal fuels. Epstein and Mantel81 reported that sub­ epithelial tumors have since been confirmed by a cutaneous injection of 0.6 mg of TEL given as four number of investigators.707273 equally divided doses to Swiss mice between birth Swiss mice fed diets containing 0.1 percent basic and 21 days of age produced malignant lymphomas lead acetate i"Pb(C2H302)2-2Pb(OH)2] developed in 1 of 26 males and 5 of 41 females, compared with both benign and malignant renal tumors.73 The same 1 of 39 males and none of 48 female control animals. compound fed to rats at the 0.1 or 1 percent level The tumors were observed 36 to 51 weeks after the similarly induced both benign and malignant kidney first injection in treated females. tumors,7074 and the incidence and size were related No definite relationship between carcinogenicity to the duration of lead feeding.74 In male Sprague- and occupational exposure to lead has been estab­ Dawley rats fed a diet containing 1 percent basic lished from human studies. In 1963, Dingwall- lead acetate, Oyasu et al.75 observed 2 cerebral Fordyce and Lane82 found only marginal evidence gliomas and 13 kidney tumors in 17 animals. for any significant incidence of malignant diseases in Van Esch and Kroes73 reported renal changes but no their study of 425 persons who had been exposed to neoplasms in 2 groups of 22 and 24 male hamsters lead while working in a battery factory. fed, for up to 2 years, a standard laboratory diet A study of the causes of mortality among lead containing 0.1 or 0.5 percent basic lead acetate. smelter and lead battery workers in 1975 concluded Renal tumors were observed in rats fed diets that the incidence of malignant neoplasms, although containing 1 percent lead acetate somewhat greater than expected, was not 76 77 [Pb(C2H302)2-3H20], and Goyer and Rhyne re­ statistically different from the incidence in the non- ported that 60 to 80 percent of rats on a diet contain­ exposed population.83 This seems to support the

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conclusion ofa Working Group ofthe International and slow recovery; (2) the more common painless Agency for Research on Cancer (IARC) that there is peripheral extensor weakness; and (3) neuropathic no evidence suggesting that exposure to lead salts and myopathic features that are indistinguishable. causes cancer in humans.69 The IARC view is sup­ This pattern is generally seen in workmen after 5 or ported by the fact that the comparable level of lead more years of chronic exposure. exposure that has been associated with malignant Patients with a history of one or more episodes of tumors in experiments on rodents is considerably acute lead intoxication develop a nephropathy higher than the toxic dose in humans.69 characterized by progressive and rather irreversible renal insufficiency. Progressive azotemia and some­ 11.3 CLINICAL LEAD POISONING times hyperuricemia are noted. Late lead nephropa­ Lead poisoning gives rise to recognized but non­ thy generally is recognized at an irreversible stage. specific syndromes including acute encephalopathy, The question of renal sequelae as a result of acute chronic encephalopathy, peripheral neuropathy, lead poisoning in children has been addressed in chronic nephropathy, and anemia. several reports. Henderson89 noted that survivors of Encephalopathy is the most severe acute clinical childhood lead poisoning in Australia demonstrate a effect of lead poisoning and may emerge rather very high frequency of chronic nephritis. Tepper,90 rapidly with the onset of intractable seizures however, did not confirm this in his Boston studies. followed by coma and cardiorespiratory arrest. Apparently, the length of exposure is of significance, When the outcome is fatal, death often occurs within as Tepper's subjects had incurred acute lead poison­ 48 hours of the onset of encephalopathy. ing during preschool years whereas the Australian In its fulminant form, development of en­ groups may have had exposure to lead for longer cephalopathy occurs in less than a week. Periods of periods of time. vomiting and apathy progressing to stupor are in­ The anemia of lead poisoning is hypochromic and terspersed with periods of hyperirritability, poor sometimes microcytic. It is also associated with memory, inability to concentrate, mental depres­ shortened red cell life span, reticulocytosis, and the sion, persistent headache, and tremor. Several re­ presence of basophilic-stippled cells. Further discus­ ports8586 indicate that children with acute en­ sion ofthe hematopoietic effects is contained in Sec­ cephalopathy may also incur acute renal injury tion 11.4. (Fanconi syndrome), showing hyperaminoaciduria, Kline studied five patients having chronic lead glycosuria, and hyperphosphaturia. poisoning.91 At autopsy, evidence was found for lead Pediatric patients with lead poisoning frequently encephalopathy in all, as well as evidence for exhibit antisocial behavior and other behavioral dis­ chronic myocarditis. The latter was characterized by orders, including loss of motor skills and speech. interstitial fibrosis with a serous exudate and They may also exhibit convulsive disorders; relatively few inflammatory cells. From these obser­ however, there are no clinical features that dis­ vations, routine electrocardiographic studies and tinguish lead-induced convulsions from other close scrutiny for evidence of myocardial damage seizure disorders. These findings are usually associ­ was recommended by the author.91 ated with blood lead levels in excess of 60 /ug/dl and Approximately 25 percent of young children who with increased density (in X-rays) at the end of long survive an attack of acute encephalopathy sustain bones (lead lines). Because the latter indicates severe permanent neurological sequelae.9294 Two prolonged absorption of lead, this clinical picture is studies9293 have indicated that a pediatric victim of termed chronic encephalopathy. The above is very acute encephalopathy has an almost 100 percent similar to the pattern seen with recurrent episodes of chance of severe permanent brain damage when acute lead poisoning with or without acute en­ returned to the same environment. cephalopathy, and the latter has been described by In its most severe form, acute lead encephalopa­ Byers and Lord87 as well as Perlstein and Attala.88 thy may be followed by cortical atrophy, hy­ However, there may have been unrecognized drocephalus ex vacuo, severe convulsive disorder, episodes of acute encephalopathy at a previous time. mental incompetence, and blindness. These results The peripheral neuropathy of lead poisoning cen­ are becoming rare, however, and subtle neurological ters on motor involvements with little effect on the deficits and mental impairment are the more com­ sensory systems. This involvement may assume three mon outcomes. clinical forms: (1) severe pain and tenderness in Many children with documented prior attacks of trunk and extremity muscles giving way to weakness symptomatic lead poisoning develop aggressive,

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hostile, and destructive behavior patterns. Although pears to be a combination of decreased erythrocyte seizure disorder and behavior abnormalities may production as a result ofthe interference oflead with diminish during adolescence, mental incompetence hemoglobin synthesis and increased destruction as a is permanent.2-87 result of direct damage by lead to the red cell itself. The specific effects of lead at various steps in 11.4 HEMATOLOGICAL EFFECTS OF LEAD erythropoiesis are discussed below. 11.4.1 Anemia Approximately 90 percent of blood lead travels 103104 Anemia of varying degree is a manifestation (often with the erythrocytes as the lead is rapidly the earliest one) of clinical lead intoxication. transferred from plasma to erythrocytes. Rosen et 100 Classically, the anemia is mildly hypochromic and al. have shown that plasma lead levels are a con­ sometimes microcytic. The anemia is associated with stant 2 to 3 figldl over a range of 10 to 150 figldl reticulocytosis (because of the shortened red cell whole blood. McRoberts,105 however, has shown survival) and the presence of basophilic stippling. that the plasma levels can fluctuate considerably Childhood lead poisoning is most frequently ob­ and are associated with the appearance of symptoms served in children 1 to 6 years old and of lower in cases of occupational exposure. Kochen104 has socioeconomic status; in both these groups the pre­ shown that erythrocytes primarily serve as a carrier valence of iron deficiency is quite high. A combina­ for blood lead, with a binding capacity well above tion of iron deficiency and increased lead intake those lead levels associated with even very heavy ex­ results in more severe anemia. Anemia, however, is posure. It would appear, then, that the whole blood also observed in children with increased lead intake content of lead is relatively independent of who are not iron deficient. Six and Goyer95 demon­ hematocrit. strated that dietary iron deficiency in rats produced increased lead retention in liver, kidney, and bone 11.4.2 Effects of Lead on Erythrocyte with increased urinary 8-ALA excretion. Kaplan et Morphology and Survival 96 al. showed that the uptake of lead by erythrocytes In lead poisoning, even in absence of iron defi­ in the presence of iron was decreased. These findings ciency, the erythrocytes are microcytic and hy­ raised the possibility that iron-deficient children are pochromic. Basophilic stippling is a frequent but in­ more susceptible to the toxic effects of lead. constant feature of lead poisoning and has been Although it is well known that anemia occurs in employed as a method of monitoring workers in the severe lead intoxication, the threshold blood lead lead industry. This test has the disadvantage of being level at which anemia occurs is not clearly estab­ nonspecific, as basophilic stippling may be observed lished. In lead workers, Sakurai97 could not demon­ in the erythrocytes of individuals with thalassemia strate any difference in hemoglobin level up to a trait and in several types of hemolytic anemia. blood lead level of 50 figldl. Tola et al.98 reported Moreover, a good correlation between the amount an effect of blood lead level on hemoglobin in a of stippled erythrocytes and blood lead level has not study of 33 workers at the beginning of their ex­ been observed.106 Recently Paglia and Valentine107 posure to lead in an occupational setting and found have indicated that the basophilic stippling in lead that after 100 days of exposure, at the time when the poisoning results from the inhibition of the enzyme average blood lead level had reached 50 figldl, the pyrimidine-5'-nucleotidase, which under normal average hemoglobin level had decreased to 13.4 g/dl conditions plays a prominent role in the cleavage of from the initial value of 14.4 g/dl (p = < 0.001). residual nucleotide chains that persist in the Pueschel99 observed a negative correlation between erythrocytes after extrusion of the nucleus. hemoglobin level and blood lead level in 40 Detreased activity of this enzyme in persons with children with blood lead levels ranging between 30 elevated blood lead levels is observed even when and 120 figldl. In this study, however, the ages ofthe basophilic stippling is not morphologically evident, individual children are not stated. A number of and it probably contributes to the shortening of the other studies also bear out the above observa­ erythrocyte survival. It is known, in fact, that a tion.100102 It is known that in children aged 1 to 6 severe chronic hemolysis is present in people who years there is a progressive physiological increase in are genetically defective in pyrimidine-5'- hemoglobin level and that both iron deficiency and nucleotidase.108 lead intoxication are most frequent irf the youngest Osmotic fragility is decreased in lead poisoning. children. This is a common feature of many microcytic The mechanism of anemia in lead poisoning ap­ anemias, as it expresses the increased surface-to-

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volume ratio that results from the reduced MITOCHONDRIAL MEMBRANE hemoglobin content of individual erythrocytes. In MITOCHONDRION lead poisoning, however, an increased osmotic resis­ tance also results from a direct effect of lead on the erythrocyte membrane because increased osmotic FERRO­ CHELATASE 09 if \ resistance may be produced by lead in vitro.- In­ SUCCINYLCoA creased osmotic resistance has been proposed as a Fo + PROTOPORPHYRIN screening test for lead poisoning in children.110 S-ALA SYNTHETASE (INCREASEI Other evidence of direct damage to the red cell Pb (DIRECTLY OR BY membrane in lead poisoning is the markedly DEREPRESSION! lowered activity of the sodium- and potassium-de­ pendent membrane ATPase, which is indispensably " 111 6-ALA COPROPORPHYRIt N coupled to active cation transport. Shortening of (INCREASE! erythrocyte survival has been shown by Hernberg et I 2 6-ALA al." using tritium-labeled difluorophosphonate DEHYDRASE and by Berk et al."3 using detailed isotopic studies (DECREASEI ofa patient with severe acute lead poisoning. Leikin PORPHOBILINOGEN (PBG! and Eng114 observed shortened survival time in Figure 11-1. Lead effects on heme biosynthesis. three out of seven children with lead poisoning and anemia. These studies indicated that hemolysis is not the exclusive mechanism of anemia and that hemoglobin, the protein that transports oxygen from diminished erythrocyte production plays an impor­ the respiratory system to every cell of the body. tant role. Hemoglobin represents 33 percent of the weight of An additional factor is a large component of in­ red cells; so a normal 70-kg male with a red cell effective erythropoiesis. This was demonstrated by mass of 3000 ml has l kg of hemoglobin, approx­ the detailed study ofthe patient of Berk et al."3 in imately 35 g of which are heme. Therefore, with a whom a marked increase of labeled stercobilin was red cell life span of 120 days, the daily production of observed after administration of labeled 14C- heme for hematopoietic use is only around 300 mg. glycine, a heme precursor. The presence of increased Lead interferes with heme synthesis at several amounts of this heme catabolite in the urine demon­ points. The two most important steps affected are the strates altered hemoglobin synthesis as a result of condensation of two molecules of 8-aminolevulinic metabolic blockage or premature intramedullary acid (8-ALA) to form the porphobilinogen ring (at destruction of red cell precursors, or both. the step catalyzed by the enzyme, 8-aminolevulinic acid dehydratase) and the insertion of iron into pro­ 11.4.3 Effect of Lead on Heme Synthesis toporphyrin IX (catalyzed by the enzyme, ferroche­ The effects of lead on heme synthesis are quite latase). Other steps in the heme synthesis are affected well known both because of their prominence and by lead, such as 8-ALA-synthetase and coprogenase; because of the large number of studies in humans these, however, may be affected indirectly through and experimental animals. The process of heme syn­ feedback derepression. thesis results in the formation of protoporphyrin IX, 11.4.3.1 EFFECTS OF LEAD ON 8-AMINO­ a complex molecule from small building blocks, LEVULINIC ACID DEHYDRATASE (8-ALAD) glycine and succinate (as succinyl coenzyme A); it AND 8-ALA EXCRETION culminates with the insertion of iron at the center of the porphyrin ring. The initial and final steps of This enzyme is highly sensitive to the effect of lead heme synthesis take place in the mitochondria, and is directly inhibited by chelation of essential SH whereas most intermediate steps take place in the groups. The inhibition may be completely reversed cytoplasm (Figure 11-1). Heme is formed in the by reactivation ofthe SH group in vitro by reducing mitochondria, and it is also an essential constituent compounds such as mercaptoethenol and of the cytochrome system located in the inner crest dithiothreitol. The observation that 8-ALAD is in­ of the mitochondria themselves and is essential to hibited by lead was first reported by Nakao et al."5 cell respiration. Besides being a constituent of the and DeBruin"6 in 1968. Hernberg et al."7 demon­ cytochrome system and of several other heme pro­ strated that the logarithm ofthe activity of 8-ALAD teins in the body, heme is the prosthetic group of was negatively correlated with blood lead level over

11-8 a range from 5 figldl (the lowest value observed) to blood, its correlation with blood lead is not surpris­ 95 figldl. In a detailed study of 25 healthy in­ ing, and 8-ALAD activity may be used to estimate dividuals with blood lead levels below 16 figldl, the blood lead with a good degree of accuracy. The in­ same investigators found a similar correlation even hibition of 8-ALAD in erythrocytes reflects a simi­ in this.lowest range."8 These data suggested the lar effect of lead in body tissues, as shown by the direct inhibition by lead of 8-ALAD with no studies of Secchi et al.120 which demonstrated that, threshold effect because the enzyme was 50 percent in 26 persons without industrial exposure to lead inactivated at a blood lead level of 16 figldl and 90 and with blood lead levels between 12 and 56 figldl, percent inactivated at a blood lead level of 55 figldl. there was a clear correlation between erythrocyte These observations have been confirmed by several and liver 8-ALAD and an expected negative cor­ other laboratories119123 for the general population, relation between blood lead and 8-ALAD in for industrial workers, and for children. In a study erythrocytes. Millar et al."9showed that when suck­ of 123 subjects, including 44 lead workers and 79 ling rats were fed diets containing lead there was a nonexposed persons (blood lead range of 4.5 to 9.3 significant and commensurate reduction of 8- figldl), Wada et al.124 noticed a similar exponential ALAD activity not only in erythrocytes but also in negative correlation between blood lead and 8- liver and brain tissues. In a recent study by Roels et ALAD. In a subsequent investigation,125 the same al.,127 however, changes in tissue ALAD and free author studied 8-ALAD in three groups: (1) 10 tissue protoporphyrin (FTP) were not found follow­ families (each including parents and one child) from ing postnatal lead administration in the rat. Lead a village far north of Tokyo with average blood lead was administered in the drinking water (0, 1, 10, 100 of 8.3 figldl (range 5 to 10), (2) 10 families from ppm) from parturition until day 21. In the offspring, central Tokyo with average blood lead of 12.8 figldl an increase in Pb-B and a reduction in ALAD ac­ (range 9 to 17), and (3) 10 male workers with tivity were found in the 10 and 100 ppm groups but average blood lead of 26.5 figldl (range 14 to 36). In no differences in hematocrit, hemoglobin, or FEP this s"tudy, a significant negative correlation between were observed. Lead storage in the kidney ofthe 100 log ALAD and blood lead was found by combining ppm group was associated with a marked rise in groups 1, 2, and 3 or groups 1 and 2. In the first kidney FTP but no differences were found in either group, however, no such correlation could be dem­ ALAD or FTP in either liver, heart, or brain. onstrated. Because this latter group comprised only The inhibition of 8-ALAD is reflected in in­ 10 families from a small village, it is possible that creased levels of its substrate, 8-ALA, in urine. failure to observe any relationship could be caused Plasma 8-ALA has been shown128 to be elevated in by the very narrow range of blood lead (5 to 10 children with severe lead poisoning; however, figldl) and/or by genetic factors. because of the technical cumbersomeness of the More recently Granick et al.126 studied the ratio techniques for measuring 8-ALA, few data are of 8-ALAD activity before and after reactivation available on 8-ALA plasma levels at lower blood with dithiothreitol in 65 children with blood lead lead levels. On the other hand, urinary 8-ALA has levels between 20 and 90 figldl. By regression been used extensively as an indicator of excessive ex­ analysis, they estimated in this series that a ratio of posure to lead, and it has even been suggested as a reactivated/nonreactivated 8-ALAD of 1 (corres­ screening tool for lead poisoning.129 Its use for this ponding to no inhibition) would occur at a blood purpose has, however, been rejected because of the lead level of 15 figldl. Because ofthe wide range of wide range of individual variability in daily excre­ variation and the small number of observations, tion observed in some studies130131 Industrial use of however, the confidence limits of their estimate are this technique has been satisfactory, however. 8 quite large. On the other hand, Hernberg et al." Several studies have indicated that an excellent have shown a negative correlation in individuals correlation exists between blood lead level and the with blood lead levels below 16 figldl, whereas the logarithm of the level of urinary 8-ALA. Selander 126 lowest blood lead level studied by Granick et al. and Cramer132 first described this relationship in was 20 figldl. For these reasons, the observations of 150 lead workers with blood lead ranging between 7 126 Granick et al. do not contradict the evidence by and 92 figldl. Their observations have been con­ 118 Hernberg et al. that 8-ALAD is already inhibited firmed by other studies123124133 in which a similar at the lowest levels of blood lead observed in correlation was observed, in one case, even in the humans in industrialized countries. Because the in­ lower blood lead level range. Selander and hibition of this enzyme is a direct effect oflead in the Cramer132 noticed that if lead workers were divided

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into two groups, those with blood lead levels below synthetase, to urinary levels of ALA at blood levels and above 40 figldl, two different linear correlation at which excretion is significant cannot be deter­ slopes could be derived, although with a lesser mined at this time. degree of correlation than the exponential relation­ ship derived from the entire group. As cited in the 11.4.3.2 EFFECTS ON IRON INSERTION IN NAS publication,2 studies from Chisholm's PROTOPORPHYRIN laboratory showed a similar exponential correlation The accumulation of protoporphyrin in the between blood lead level and urinary 8-ALA is 51 erythrocytes of humans with lead intoxication has children aged 1 to 5 having blood lead levels rang­ been known since the 1930's.135 Its use as an indica­ ing between 25 and 75 figldl. In 55 adolescents with tor of lead body burden, however, has been limited blood lead levels ranging from 8 to 40 figldl, by the technical difficulties associated with the however, no clear correlation could be observed. It measurement of protoporphyrin by solvent partition appears that apart from this last observation (which and spectrophotometry. In 1972, the development is restricted to adolescents, the great majority of of a simpler and more accurate technique, combin­ whom had blood lead levels in a very narrow range: ing simplified extraction and fluorometry, made the 14 to 24 figldl), all other studies reported show a measurement of protoporphyrin a widely used and clear exponential increase in 8-ALA urinary excre­ accessible test.136 As discussed in Chapter 9, several tion with increase in blood lead. These observations modifications of this technique have been parallel the reported exponential inhibition of the developed, including an instrument that measures enzyme 8-ALAD and indicate that this is one ofthe protoporphyrin by direct fluorescence in capillary earliest effects of lead on heme synthesis. blood samples without any extraction or manipula­ The urinary excretion of 8-ALA does not exceed tion of the blood sample. the normal range (0.6 figldl) until the blood lead Accumulation of protoporphyrin in the level reaches 40 figldl. The normal range, however, erythrocytes is the result of decreased efficiency of is derived from values obtained from individuals iron insertion into protoporphyrin, the final step in with blood lead levels up to 40 figldl. It is apparent heme synthesis, which takes place inside the that if 8-ALAD were inhibited by lead without any mitochondria. When this step is blocked by the effect threshold of concentration and if 8-ALA were of lead, large amounts of protoporphyrin without similarly affected, the definition of a normal range iron accumulate in the erythrocyte, occupying the of 8-ALA excretion for individuals with a blood available heme pockets in hemoglobin. Hence, pro­ lead level less than 40 figldl would be ambiguous at toporphyrin, rather than heme, is incorporated in best. Some of the discrepancies reported in the the hemoglobin molecule where it remains literature could in part reflect the larger variability throughout the erythrocyte life span (120 days). of 8-ALA urinary excretion in comparison with The accumulation of protoporphyrin in lead erythrocyte 8-ALAD. In a detailed study by Alessio poisoning is different from that observed in 123 et al. of 169 males with blood lead levels ranging erythropoietic protoporphyria, a congenital dis­ from 5 to 150 figldl, the correlation between blood order in which excess protoporphyrin is produced lead and 8-ALAD was much greater than with urin­ after heme synthesis is complete. In that case, the ex­ ary 8-ALA. For these reasons and because ofthe un­ cess of protoporphyrin formed (as a result of a con­ certainty of defining a normal range, it is generally genital defect in ferrochelatase) is attached to the accepted that urinary 8-ALA becomes clearly ab­ surface of hemoglobin at a site that bridges the a- normal at blood lead levels greater than 40 figldl. It and f3-chains of hemoglobin.137138 Because this type has been postulated that there may be an excess of 8- of bond to hemoglobin is very loose in ALAD activity, so that normal 8-ALA metabolism erythropoietic protoporphyria, protoporphyrin is still sustained by even 50-percent-inhibited diffuses through the plasma into the skin where it in­ enzyme at blood lead levels near 40 figldl. Above duces photosensitivity. In lead intoxication, on the this value, however, the inhibition results in func­ other hand, the protoporphyrin in hemoglobin is tional impairment and clear accumulation of 8- bound more firmly to the heme pocket; hence, no ALA, and increased urinary excretion may be ob­ diffusion into the plasma occurs and no photosen­ served. A recent study suggests that ALA may, in sitivity is observed, despite extremely elevated fact, be toxic systemically. The relative contribu­ erythrocyte protoporphyrin levels. tions from decreased utilization of ALA, as a result An additional important difference between the of ALAD inhibition and the derepression of ALA- increased protoporphyrin level in the erythrocytes

11-10 of persons with lead intoxication and erythropoietic of lead is exerted on the enzyme itself or on overall protoporphyria is the fact that only in the former is mitochondrial function. It is possible that the center ofthe protoporphyrin molecule occupied mitochondrial transport of iron and ferrochelatase by zinc.'39 This difference is probably caused by the are both affected by lead. different affinity for zinc of protoporphyrin in the The effect of lead on iron incorporation into pro­ heme pocket. In lead intoxication, then, the largely toporphyrin is not limited to the normoblast and/or prevalent species is zinc protoporphyrin, whereas in to the hematopoietic system. Formation ofthe heme- erythropoietic protoporphyria it is an unchelated containing protein, cytochrome P450, which is an in­ protoporphyrin base. These two compounds differ in tegral part of the liver mixed-function oxidase fluorometric spectra and the two conditions may be system, may also be inhibited by lead.144 Accumula­ 140 easily distinguished by spectrofluorometry. Zinc tion of protoporphyrin in the presence of lead has protoporphyrin, attached in the heme pocket of been shown to occur also in cultured cells of chick hemoglobin, is also the prevalent species observed in dorsal root ganglion, indicating that inhibition of iron deficiency, another condition in which an in­ heme synthesis takes place in the neural tissue as creased level of protoporphyrin is observed in the well.145 These observations, and the fact that lead is erythrocytes. known to disrupt mitochondrial structure and func­ Accumulation of protoporphyrin in the tion, indicate that the lead effect on heme synthesis is erythrocytes in lead poisoning indicates a failure of exerted in all body cells, possibly with different the last step of heme synthesis. This could result dose/response curves holding for effects in different either from a direct effect of lead on ferrochelatase cell types. On the other hand, it must be noted that itself or from an effect of lead on mitochondrial increased levels of protoporphyrin in the membranes of erythroid tissue in bone marrow, with erythrocyte reflect an accumulation of substrate and consequent failure of iron transport. The latter therefore imply a functional alteration of mechanism would make iron, one of the two sub­ mitochondrial function in the same way that the in­ strates of ferrochelatase, less available to enzyme ac­ creased urinary excretion of urinary 8-ALA implies tion, with subsequent accumulation ofthe unutilized impairment. In other words, if a reserve activity of substrate, protoporphyrin IX. ferrochelatase exists, such as has been suggested for Interference by lead with the mitochondrial 8-ALAD, accumulation of protoporphyrin in the transport of iron in the normoblast appears to be the erythrocytes indicates that this has been hampered most likely mechanism underlying the increased by the lead effect to the point that the substrate has level protoporphyrin in the erythrocytes. Four facts accumulated. For these reasons, as well as for its im­ support this statement: (1) iron accumulation within plication of the impairment of mitochondrial func­ the erythrocyte is diminished by the presence of tion, accumulation of protoporphyrin has been taken to indicate physiological impairment relevant to lead, whereas iron incorporation into heme is com­ 146 pletely inhibited; (2) lead is deposited on the human health. mitochondrial membrane, where it produces pro­ The elevation of erythrocyte protoporphyrin was found ultrastructural changes; (3) iron transport shown to be exponentially correlated with blood through the mitochondrial membrane is ac­ lead level by Piomelli in a study of 90 children, complished by both energy-dependent and energy- covering the blood lead level range from 5 to 90 independent mechanisms that are impaired by /ig/dl.147 In a later study of 1038 children, 568 of 141 lead; and (4) in iron deficiency (when fer­ whom had blood lead levels greater than 40 rochelatase activity is normal but iron is scarce) zinc /tg/dl,148 this correlation was confirmed, and it was protoporphyrin bound in the heme pocket is ac­ clearly shown that all children with blood lead cumulated, whereas in erythropoietic pro­ levels greater than 60 figldl had erythrocyte pro­ toporphyria (when iron is normal but ferro­ toporphyrin greater than 250 figldl red blood cells 142143 chelatase activity is decreased ) free pro­ (RBC's). Kamholtz et al.149 and Sassa et al.150 also toporphyrin base loosely attached to the hemoglobin showed a similar degree of correlation and indicated surface is formed. that a value of 140 fig FEP/dl RBC's would appear Experimental evidence from animal studies and to be a more appropriate cut-off point for screening epidemiologic human studies, using intact children for lead poisoning. This value, also sug­ mitochondria, have demonstrated the failure of iron gested by McLaran et al.,151 was accepted by incorporation into protoporphyrin in the presence of Piomelli et al.,152 who indicated that more than 70 lead. These studies cannot clarify whether the effect percent of children with a blood lead level of 40 to

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49 figldl have erythrocyte protoporphyrin in excess focused on persons (children or adult workers) with of this value. Several additional studies have con­ markedly elevated blood lead. Some studies, firmed the exponential correlation between blood however, have shed light on the threshold level lead and erythrocyte protoporphyrin in chil- below which no effect is observed. In a study of 123 133 140 157 158 dreni40,i53-i56anciinleadworkers. ' ' ' . children with blood lead levels over the range of 20 150 150 to 40 figldl, Sassa et al. could not detect any Sassa et al. demonstrated that a better correla­ 159 tion was observed between blood lead and threshold effect. Data from Roels et al., who erythrocyte protoporphyrin in children with a steady studied 143 school children having blood lead levels blood lead level. This finding suggested that a sig­ ranging from 5 to 40 figldl, indicate a threshold effect at blood lead levels between 15 and 20 figldl. nificant part ofthe scatter observed when blood lead 169 is correlated to erythrocyte protoporphyrin on a In a study by Piomelli et al. of 1816 children aged random basis is the result of fluctuations of lead 2 to 12 years (median age 4.7 years), the threshold caused by day to day variation and experimental er­ for no effect of blood lead on erythrocyte proto­ ror. porphyrin was estimated to be 15.5 figldl, using both probit analysis and segmental curve-fitting tech­ 140 Lamola et al. demonstrated that the slope of niques. elevation of erythrocyte protoporphyrin versus Because an elevation of erythrocyte pro­ blood lead is steeper in children than in adult lead toporphyrin is caused also by iron deficiency, it is workers. This observation was confirmed by Roels important to take into consideration the iron state of et al.159 who also demonstrated that the slope of the population under study in any evaluation ofthe elevation is similar in children and females. Reigert relationship of this hematological index to lead ex­ et al.160 and Levi et al.161 also demonstrated that an posure. No information is available with regard to elevation of erythrocyte protoporphyrin can predict the iron state of the population studied by Sassa et which children tend to increase their blood lead 150 159 al. In the Roels study, similarly, no direct level and suggested that erythrocyte protoporphyrin measurements of the iron status were obtained; is a more valuable indicator of childhood body however, the children studied ranged in age from 10 burden of lead than the blood lead level itself. In to 15 years, a group in which iron-deficiency anemia adult workers, the elevation of erythrocyte pro­ is uncommon. Moreover, the differences in blood toporphyrin was shown to correlate with blood lead lead were clearly related to living near or away from level, ALAD, ALA-U, and the duration of exposure lead-emitting smelters. There is no reason to believe to lead.158 Chisholm et al.162 suggested that in that the children who live near a smelter should have children erythrocyte porphyrin is a better indicator lower iron stores than the children who live in rural of overexposure to lead than blood lead. In addition areas. Also, in the same study, it must be noted that to being elevated in lead intoxication, erythrocyte the hematocrit ofthe children living in the rural area protoporphyrins may also be elevated in iron defi­ was slightly but significantly lower than the ciency, but to a lesser degree. Several studies have hematocrit of the children living near the smelter; indicated that erythrocyte protoporphyrin levels are therefore, if anything, the prevalence of any iron an excellent indicator of the body iron store164165 deficiency may have been greater in the rural and that these levels may also be used to discrimi­ children (with the lowest EP) than in the children nate between the microcytic anemia of iron deficien­ living near the smelter (with the highest EP). These cy (where they are elevated) and of thalassemia trait facts suggest that iron-deficiency anemia was not a (where they are normal).166"168 factor in the elevation of erythrocyte protoporphyrin These observations and the data collected on over observed in this study, but that this EP increase was 300,000 children screened by both erythrocyte pro­ directly related to lead. In the study of Piomelli et toporphyrin and blood lead in New York City were al.,169 an analysis of children aged 2 to 4 years versus the basis for the statement by the Center for Disease children older than 4 years failed to show any Control (CDC) in which an elevation of erythrocyte difference in the EP/blood lead relationship. This protoporphyrin above 60 figldl of whole blood in indicates that iron-deficiency anemia, which is much the presence of a blood lead level above 30 figldl more prevalent in the younger children, did not in­ were indicated as cut-off points for the detection of 146 fluence the EP response. Moreover, in the same childhood lead poisoning. study, the iron stores were measured in children with Most studies on the relationship between blood lead levels < 15 figldl and in children with erythrocyte protoporphyrin and blood lead have blood lead levels of 15 to 28 figldl and no difference

11-12 was observed. It appears, therefore, that the effect of 11.4.5 Summary of Effects of Lead on the the lead on EP, which is extremely well documented Hematopoietic System at the much higher blood lead level, occurs also in A number of significant effects on the children with blood lead levels between 15 and 28 hematopoietic system in humans have been observed figldl. in lead poisoning. These effects are prominent in These studies consistently demonstrate that an clinical lead poisoning, but they are still present to a elevation of erythrocyte protoporphyrin, which indi­ lesser degree even in persons with lower body bur­ cates physiological impairment of heme synthesis dens of lead. and mitochondrial function, can be detected in Anemia is a clinical fixture of lead intoxication. It children at a blood lead level that is well below results from both increased erythrocyte destruction levels normally encountered in screening pro­ and decreased hemoglobin synthesis. Erythrocytes cedures. are microcytic and have abnormal osmotic fragility as a result of direct effect of lead on the cell 11.4.4 Other Hematological Effects membrane, and show basiphilic stippling caused by the inhibition of pyrimidine-5'-nucleotidase. The effects of lead on 8-ALAD and on iron incor­ Erythrocyte survival time is shortened, and this poration in protoporphyrin are the best known. In results in hemolysis. lead intoxication, however, other abnormalities of heme synthesis are observed. These include an in­ In children, a threshold level for anemia is about creased activity of 8-ALA synthetase,170 which may 40 fid Pb/dl, whereas the corresponding value for result by derepression, according to the scheme of adults is about 50 fig Pb/dl. negative feedback control proposed by Granick and Lead interferes with hemoglobin synthesis by in­ Levene.171 In vivo inhibition of coproporphyrinogen hibiting synthesis of the globin moiety and affecting and uroporphyrinogen decarboxylases in rabbits'72 several steps in the synthesis of the heme molecule. and inhibition of uroporphyrinogen I synthetase173 Most sensitive to lead in the heme synthetic pathway have been reported. On the other hand, no ac­ is the activity of the enzyme 8-ALAD, a zinc-acti­ cumulation of porphobilinogen has been observed in vated enzyme that mediates the conversion of two humans. An increased excretion of coproporphyrin molecules of 8-ALA into prophobilinogen. Inhibi­ in the urine of lead workers and children with lead tion of this enzyme results in increased plasma levels poisoning is well known. Urinary coproporphyrin and urinary excretion of 8-ALA. Lead also inhibits has been used extensively as a clinical indicator of the last step (incorporation of iron into pro­ lead poisoning. It is not known, however, whether toporphyrin), which takes place in the mitochondria, this effect results from specific enzyme inhibition, probably by interference with the mitochondrial from upstream accumulation of substrate secondary transport of iron and coproporphyrin. This effect to inhibition of iron incorporation into proto­ results in the accumulation of coproporphyrin, porphyrin, or from both; or, alternatively, whether it which is excreted in the urine, and of pro­ is expressed as a disturbance of coproporphyrin toporphyrin, which is retained in the erythrocytes, in transport through the mitochondrial membrane. the heme molecule. The overall effect of lead is a net Similarly, no data are available to establish a decrease in heme synthesis, which, in turn threshold blood lead level below which no excess derepresses the enzyme involved in the first step of coproporphyrin excretion in the urine takes place. heme synthesis, 8-ALA synthetase. Besides the effect of lead on heme synthesis, Inhibition of 8-ALAD occurs at extremely low hemoglobin synthesis may also be impaired because blood lead levels and has been shown to start at a of inhibition by lead of the synthesis of globin (the blood lead level of 10/ig/dl. The resultant increased protein moiety of hemoglobin). Kassenar et al.174 urinary 8-ALA excretion also starts at a very low showed impairment of globin synthesis. This work blood lead level and becomes pronounced at a blood was confirmed by the results of Wada et al.170 White lead level 240/ig/dl. and Harvey175 showed a decreased synthesis of a The precise threshold for coproporphyrin excre­ chains compared to /3-globin chains. Recently, Ali tion is not well established. It is probably similar to et al.176 have shown an effect on globin synthesis in the threshold for 8-ALA, but it is less specific. An vitro on human reticulocytes at lead concentrations increase in erythrocyte protoporphyrin occurs at a as low as IO6 M, which corresponds to a blood lead threshold blood lead level of approximately 16 level of 20/ig/dl. /ig/dl in children. In adult females, the threshold is

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probably similar. In adult males, the threshold is duce much less well-defined but medically signifi­ probably slightly higher (20 to 25 /ig/dl). The cant neurobehavioral deficits in apparently threshold for increase in 8-ALA synthetase is not es­ asymptomatic adults and children, that is, in the ab­ tablished, but increases have been noticed at blood sence of the neurological symptoms or other signs lead levels of 240 /ig/dl. that typify acute lead intoxication requiring immedi­ Although doubt exists as to the health-effects sig­ ate clinical treatment. nificance of 8-ALAD inhibition, increased urinary The range of lead exposures necessary to produce 8-ALA excretion above 40 figldl is accepted as an the more subtle, subclinical neurobehavioral deficits effect probably reflecting physiological impair­ is difficult to estimate with certainty and remains a ment.2 Elevation of erythrocyte protoporphyrin has matter of considerable controversy. There is some the same implication of physiological impairment in evidence reviewed below that suggests that such vivo as is found in urinary 8-ALA. Also, because effects may occur at blood lead levels even as low as elevation of erythrocytic protoporphyrin indicates 30 to 40 /ig/dl, whereas certain other negative find­ impairment of mitochondrial function, it is con­ ings suggest the lack of neurobehavioral effects at sidered of greater physiological relevance.177 For blood lead levels less than 80 /ig/dl. In an effort to these reasons, the consensus of clinicians who partic­ estimate the exposure levels necessary for manifesta­ ipated in the preparation ofthe statement in 1975 by tion of the full range of neurobehavioral effects of CDC together with the American Academy of lead, the present discussion will critically review the Pediatrics was that this finding should be used as an literature dealing with the obviously toxic effects of indicator of a significant and worrisome body high level lead exposures and with the more subtle burden of lead. neurobehavioral effects associated with lower ex­ posure levels. 11.5 EFFECTS OF LEAD ON NEURO­ The relevant literature on the neurobehavioral PHYSIOLOGY AND BEHAVIOR effects of lead has been derived from studies of both Neurological and behavioral deficits have long humans and other mammalian species. Such effects been recognized as some of the more severe conse­ have been indexed by means of a variety of ap­ quences of toxic exposure to lead.178182 What levels proaches, including: (1) the assessment of structural of lead exposure are necessary to produce specific neuropathology by classical histological and deleterious neurological or behavioral effects and ultrastructural analyses of morphological damage; whether such effects are reversible, however, have (2) the analysis of altered neurochemical parameters been controversial medical issues extensively de­ or processes by various biochemical assays; (3) the bated since the early 1900's. M uch of the impetus for assessment of altered electrophysiological responses debate on the subject has been generated by in both the central and peripheral nervous system; progressively increasing medical concern over an (4) the assessment of neurobehavioral effects both by evolving scientific literature that has consistently neurological examinations and diverse types of suggested, as more information is gained, that lead behavioral testing methods; and (5) the assessment exposure levels previously accepted as harmless are of alterations in neuropharmacological responses actually sufficient to cause significant neurological affecting many of the types of variables assessed by or behavioral impairments. At present it is generally the other approaches. The effects of toxic, high-level accepted that, at toxic, high levels of lead exposure exposures to lead have been well documented by that produce blood lead levels greater than 80 to most ofthese approaches. At lower-level exposures, 100 /ig/dl, a person is at unacceptable risk for the however, the demonstration of lead effects by any of occurrence of the clinical syndrome of fulminant the above types of assessments has been complicated lead encephalopathy. This syndrome includes by several other methodological considerations that neurological and other symptoms of such severity should be noted as a prelude to any critical review of that immediate medical attention and, frequently, the literature. hospitalization is demanded in order to avoid ir­ Data about lead exposure have been obtained via reversible neural damage or death. The risk in­ two basically different methods, epidemiological volved is unacceptable because of the unpredic­ and experimental. Unfortunately, these tability ofthe symptoms observed at high blood lead methodological techniques are highly correlated levels. Based on the literature reviewed below, with the species studied; that is, epidemiological it now also appears that lower levels of lead ex­ posure, yielding blood levels below 80 /ig/dl, pro­ techniques, with their unique problems, provide most human data, and experimental techniques are

11-14 used to provide most nonhuman data. Although the effects of lead on the nervous system include (1) epidemiological studies have immediate environ­ the duration and intensity of exposure and (2) age at mental relevance at the human level, there are often exposure. In reference to age at exposure, evidence difficult problems associated with interpretation of exists for greater vulnerability of the developing the findings. With epidemiological studies, the exact nervous system in the young than of the fully parameters of the most recent exposure level, the matured nervous system in adults. Particular atten­ duration of any level of exposure, and the mode of tion will, therefore, be accorded to the discussion of intake usually cannot be entirely known. Similarly, the neurobehavioral effects of lead in children as a any previous lead levels and exposure durations special group at risk. usually cannot be definitively established. It is also possible that other variables that were highly corre­ 11.5.1 Human Studies lated with the presence of environmental lead, such 11.5.1.1 EFFECTS OF HIGH-LEVEL LEAD as socioeconomic level, previous behavioral or EXPOSURES neurological damage, etc., are responsible for the The severely deleterious effects of exposures to effects observed rather than lead exposure alone. high levels of lead, especially for prolonged periods There is a need for appropriate and sensitive that produce overt signs of acute lead intoxication, measures of exposure effects, especially when testing are by now well documented in both adults and for low-level effects for which less-than-dramatic children. The most profound effects that occur in neurobehavioral deficits can be expected. Neverthe­ adults are referred to as the clinical syndrome of less, the contribution of lead exposure to any lead encephalopathy, described in detail by neurobehavioral deficit(s) can be reasonably esti­ I83186 numerous investigators. Early features of the mated with proper controls for many of the above syndrome that may develop within weeks of initial extraneous factors. exposure include dullness, restlessness, irritability, Obviously such parameters as exposure levels and poor attention span, headaches, muscular tremor, durations can be defined with much more precision hallucinations, and loss of memory. These symptoms in experimental studies carried out in the laborato­ may progress to delirium, mania, convulsions, ry. Unfortunately, however, appropriate experimen­ paralysis, coma, and death. The onset of such serious tal designs are frequently lacking. In addition, en­ symptoms can often be quite abrupt, with convul­ vironmental relevance of experimental laboratory sions, coma, and even death occurring very rapidly data is limited by two major considerations. The in patients that shortly before were apparently most serious of these is the fact that nonhuman asymptomatic or exhibited much less severe models are, of necessity, typically used for the estab­ symptoms of acute lead intoxication.I85187 lishment of dose-response curves, and it is well Symptoms of encephalopathy similar to those that known that a large species difference exists in sen­ occur in adults have been reported to occur in in­ sitivity to lead,52 so that adequate nonhuman ex­ fants and young children,8590'85188189 with a posure models are difficult to devise. A second markedly higher incidence of severe en­ problem is that animal experiments frequently use cephalopathic symptoms and deaths occurring in doses of lead much higher than would be expected to them than in adults. This may reflect the greater occur in the environment. Besides the question of ex­ difficulty in recognizing early symptoms in young posure levels, experimenters must attend to proper children that allows intoxication to proceed to a experimental controls for possibly reduced nutrition more severe level before treatment is initiated. In levels because of food palatability, if the delivery regard to the risk of death in children, the mortality system is via food or water; effects of altered mater­ rate for prechelation therapy period encephalopathy nal behavior, if the delivery is via the mother's milk cases was approximately 65 percent.190 Various or the placenta, etc. Further, if central nervous authors have reported the following mortality rates system (CNS) alterations are noted, it is often for children experiencing lead encephalopathy since difficult to separate damage caused by direct versus the inception of chelation therapy as the standard indirect effects on neural tissue. Again, despite the treatment approach: Ennis and Harrison,191 39 per­ above difficulties, useful data on the cent; Agerty,192 20 to 30 percent; McKhann and neurobehavioral effects of lead have been obtained Vogt,189 24 percent; Mellins and Jenkins,193 24 per­ through animal studies, with potential implications cent; Levinson and Zeldes,194 19 percent; Tanis,195 for understanding human exposure effects. 18 percent; and Lewis et al.,19 5 percent. These data, 2 Key variables that have emerged in determining as well as other data tabulated more recently, indi-

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cate that once lead poisoning has progressed to the myelin membranes.201 Remyelination has been ob­ point of encephalopathy a life-threatening situation served in animal studies, suggesting either that the clearly exists and, even with medical intervention, is lead effect may be reversible or that not all of the apt to result in a fatal outcome. Schwann cells are affected equally.201 Reports of pes The morphological findings in cases of fatal lead cavus deformities resulting from old peripheral encephalopathy vary.'97*'99 On macroscopic ex­ neuropathies in humans,202 however, suggest that amination the brains are often found to be lead-induced neuropathies of sufficient severity edematous and congested. Microscopic findings of could result in permanent peripheral nerve damage. cerebral edema, altered capillaries (endothelial hy­ Morphologically, the neuropathy is charac­ pertrophy and hyperplasia), and a perivascular glial teristically detectable only after prolonged or high proliferation are often noted. Neuronal damage is exposure to lead or both; data from experimental variable and may be caused by anoxia. In some cases studies indicate that there are distinctly different gross and microscopic changes are minimal.198 The sensitivities among different species. neuropathologic findings as reported are essentially Perhaps of even greater concern than the occur­ the same for adults and children. Lead encephalopa­ rence of fatalities are the neurological sequelae that thy is considered by some to be primarily a occur in cases of severe or prolonged nonfatal vasculopathy, with the encephalopathy reflecting episodes of lead encephalopathy that are perturbed blood-brain barrier function;198199 that qualitatively quite similar to those seen with many is, damage to neuronal elements may be secondary types of traumatic or infectious cerebral injury, with to lead effects on the vascular system. Evidence for the occurrence of permanent sequelae being more such effects has been advanced by Pentschew.198 common in children than in adults.8590193 The most Pentschew198 described neuropathology findings severe sequelae in children are cortical atrophy, hy­ for 20 cases of acute lead encephalopathy in infants drocephalus, convulsive seizures, and severe mental and young children. The most common finding was retardation.8590193 More subtle sequelae also occur, activation of intracerebral capillaries characterized such as impaired motor coordination, altered senso­ by dilation ofthe capillaries with swelling ofthe en­ ry perception, shortened attention span, and slowed dothelial cells. Diffuse astrocytic proliferation in the learning. These latter effects have been reported in gray and white matter was also present. According children with known high exposures to lead but to Pentschew, this proliferation is the earliest without a history of the life-threatening forms of morphological response to an increase in per­ acute encephalopathy.85T86-9''203 Of historical in­ meability of the blood-brain barrier (dysoria). terest here in relation to the extremely slow progress Concurrent with the dysoric alterations were in recognizing the full consequences of lead intox­ changes that Pentschew198 attributed to ication is the fact that, although many cases of child­ hemodynamic disorders. These ischemic changes hood lead poisoning had been reported since the were manifested as cell necrosis, perineuronal in­ early 1900's,'80*'82 it was several decades before the crustations, or neuronophagia (loss of neurons). The work of McKhann and Vogt189 and Byers and isocortex and the basal ganglia were areas of pre­ Lord85 called attention to the long-term irreversible dilection for the ischemic changes. Pentschew con­ neurobehavioral sequelae of acute lead intoxication. cluded that the structural changes in infantile lead Establishing precise threshold values for lead ex­ encephalopathy are a mixture of dysoric and posures necessary to produce the above acute intox­ hemodynamic parenchymal alterations. In the ication symptoms or sequelae in humans is difficult cerebellum, which in a restricted sense is the pre­ in view ofthe usual inaccessability of extensive data dilection area of damage, the changes are purely on environmental lead levels contacted by the vic­ dysoric. tim, the period of exposure, or the body burdens of In addition to producing the above effects on the lead existing prior to the manifestation of clinically CNS, lead also clearly causes damage to peripheral significant symptoms. Nevertheless, enough infor­ nervous systems (PNS) of both man and animals at mation is available to allow for reasonable estimates toxic, high exposure levels. The PNS changes in­ to be made regarding the range of blood lead levels volve predominantly the large myelinated motor needed to produce acute encephalopathic symptoms fibers.200 Pathologic changes in the PNS consist of or death. According to Kehoe204206 blood lead segmental demyelination and in some fibers, axonal levels well in excess of 120 /ig/dl are usually necess­ degeneration.200 The lead effect appears to be in the ary to produce such deleterious irreversible effects Schwann cell, with concomitant disruption of the for adults. Recurrent bouts of lead intoxication in

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the absence of acute encephalopathy may also lead ports purportedly demonstrated acute encephalopa­ to progressive mental deterioration. Other data ex­ thy in children at blood lead levels even down to 30 ist, however,187 that suggest that acute lead intoxica­ to 50 /ig/dl. Detailed analyses of the articles tion, including severe gastrointestinal symptoms or referenced, however, indicate that the actual data signs of encephalopathy or both, can occur in adults reported in most did not clearly associate such low- at lead levels somewhat less than 100 /ig/dl; but am­ level exposures to the occurrence of acute en­ biguities in these data make interpretation difficult. cephalopathy symptoms. Still, cases in at least some The data on threshold levels for children indi­ of the referenced articles and in other reports cate that lower blood lead levels have been asso­ reviewed below suggest that acute encephalopathy ciated with the occurrence of acute encephalopathy occurred in a few children at blood lead levels symptoms and death. Probably the most extensive below 100 /ig/dl. Again, the ambiguities in these compilation of information bearing on this point is a data regarding confirmation of lead exposure and summarization187 of data from the work of elimination of alternative etiological factors make Chisholm84207 and Chisholm and Harrison.90 That interpretation difficult. A further precaution has to data compilation relates the occurrence of acute en­ do with the analytical methods themselves in terms cephalopathy and death in children in Baltimore to of using good methods with skilled personnel. blood lead levels determined by the Baltimore City In 1938, Gant208 reported on five cases of acute Health Department (dithizone method) between lead encephalopathy in children under 2 years of 1930 and 1970. Elevated blood lead levels associ­ age. Blood lead levels of 60 and 80 /ig/dl, as well as ated with asymptomatic cases or less severe signs of 190, 240, and 320 /ig/dl, were obtained for the acute lead poisoning were also tabulated. different children upon first admission to the hospi­ Asymptomatic increased lead absorption was ob­ tal. All five had convulsions. Smith187 listed a 3- served at blood levels ranging from 60 to 300 /ig/dl year-old female patient as having a blood lead level (mean = 105 figldl). Acute lead poisoning of 60 /ig/dl at the time of acute lead intoxication symptoms, other than signs of encephalopathy, were from paint ingestion, followed by death attributed to observed from approximately 60 to 450 /ig/dl (mean plumbism 2 days after a 70 /ig/dl reading was ob­ = 178 /ig/dl). Signs of mild encephalopathy (hy- tained during a period when only mild symptoms of perirritability, ataxia, convulsions) and severe en­ lead poisoning were present. In 1956, Bradley et cephalopathy (stupor, coma, convulsions repeated al.209 reported that 19 children under 5 years old over a 24-hr period or longer) were associated with from a low income area of Baltimore were found to blood lead levels of approximately 90 to 700 or 800 show CNS symptoms that included irritability, /ig/dl, respectively (means = 328 and 336 /ig/dl, lethargy, or convulsions at blood lead levels below, respectively). The distribution of blood lead levels as well as above, 100 figldl. Eight children who had associated with death (mean = 327 /ig/dl) was es­ been previously classified as asymptomatic and who sentially the same as for levels yielding either mild had blood lead levels of 50 to 80 /ig/dl were later or severe encephalopathy. These data suggest that hospitalized during the study210 for treatment of threshold blood lead values for death in children are acute lead encephalopathy; blood lead levels at the essentially identical to those for acute encephalopa­ time of later hospitalization were not reported but thy and that such effects are manifested in children were likely further elevated. Other data are re­ starting at blood lead levels of approximately 100 ported2" on 10 children from a low-income area of /ig/dl. Other evidence reviewed below, however, Providence, Rhode Island who were selected at 4 to suggests that the threshold for acute encephalopathy 8 years of age for a follow-up investigation of possi­ effects in the most highly susceptible children may, ble long-term neurobehavioral deficits resulting in some rare instances, be somewhat lower than the from earlier acute encephalopathy episodes. Blood 100 /ig/dl figure arrived at on the basis of the lead assays obtained at the time of the initial hospi­ Baltimore data compilation presented above. talization of the children because of acute en­ Occasionally appearing in the literature since the cephalopathy symptoms (4 out of 10 had convul­ 1930's are scattered reports of acute lead encepha­ sions, 8 out of 10 had ataxia, 7 out of 10 had drowsi­ lopathy or death occurring in children at what ness, and 6 out of 10 had irritability) yielded max­ were formerly considered to be moderately elevated imum blood lead values that averaged 88 ± S.D. 41 blood lead levels. For example, Cumings185 listed /ig/dl. Because individual cases were not described, references to studies on acute lead encephalopathy however, no clear association between particular that appeared from 1938 to 1956. Several ofthe re­ lead intoxication symptoms and specific blood lead

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levels can be established. Overall, the above reports neurologically related symptoms among occupa­ suggest that at least some children — perhaps tionally exposed lead workers. Some recent especially inner-city children less than 4 years old — epidemiological studies have investigated possible may be vulnerable to acute encephalopathy at blood relationships between moderately elevated blood lead levels of 80 to 100 /ig/dl. lead levels and general health as indexed by records From the preceding discussion, it can be seen that of sick absences that have been certified by physi­ severity of symptoms varies widely for different cians. No correlation between elevated blood lead adults or children as a function of increasing blood levels and sickness rates or types of symptoms re­ 212 lead levels. Some show irreversible CNS damage or ported were found for groups of workers in a lead death at levels less than 100 /ig/dl, whereas others storage battery factory from high-, medium-, and may not show any of the usual clinical signs of lead low-exposure areas versus control workers in nonex- intoxication even at blood lead levels in the 100 to posure areas of the same plant. It should be noted, 200 /ig/dl range. This difference may be caused (1) however, that mean blood lead levels for workers in by individual biological variation in susceptibility to the three exposure groups were 60, 50, and 42 /ig/dl, lead effects; (2) by changes in blood lead values respectively, compared with 45 /ig/dl for the so- from the time of initial damaging intoxication; (3) by called nonexposure control group, rendering the better tolerance for a gradually accumulating lead conclusions of the report of dubious value. Similar 213 burden; or (4) by any number of other interacting negative findings were reported by Robinson for factors, such as nutritional state or inaccurate deter­ tetraethyl-lead (TEL) workers having mean blood minations of blood lead. In any case, in attempting lead values of 43 /ig/dl and daily urinary excretion to estimate exposure levels for adverse health effects of 0.089 mg of lead per liter urine over an 8- to 10- of lead, the range of exposure levels yielding damag­ year period (3 to 4 times the rate for control group). ing effects to the most susceptible individuals needs Data on sickness rates were based on a retrospective to be emphasized rather than any average level at study of records over a 20-year period. Absence or which such effects are seen. For adults, it would ap­ sickness reports, however, are probably not sensitive pear that the most susceptible individuals do not ex­ enough measures to detect subtle neurobehavioral hibit acute encephalopathy symptoms until blood symptoms. lead levels of 100 /ig/dl are reached or, more Only a few studies have employed more sensitive typically, are substantially exceeded. In regard to psychometric and neurological testing procedures in children, the majority of cases showing acute en­ an effort to demonstrate subclinical lead-induced cephalopathic symptoms have blood lead levels of neurobehavioral effects in adults. For example, 100 /ig/dl or more. For a very few cases, levels as Morgan and Repko214 reported preliminary results low as 80 /ig/dl have been reported. of an extensive study of behavioral functions in 190 lead-exposed workers (mean blood lead level = 11.5.1.2 EFFECTS OF LOW-LEVEL LEAD EX­ 60.5 ± 17.0 /ig/dl). In 68 percent of the subjects, POSURES blood lead was <80 /ig/dl. The majority ofthe sub­ Also of great relevance for establishing safety jects were exposed between 5 and 20 years. The limits for exposure to lead is the question of whether authors examined 36 nonindependent measures of exposures lower than those producing symptoms of general performance and obtained 44 measures of overt acute intoxication may exert more subtle, sensory, psychomotor, and psychological functions. subclinical neurobehavioral effects in apparently Initial data analysis suggested that blood lead levels asymptomatic adults or children. Attention has been correlated with several reaction-time measures, and focused in particular on whether exposures leading 8-ALAD changes correlated with effects on hand- to blood lead levels in the 30 or 40 to 80 /ig/dl range eye coordination. This study, therefore, suggested may lead to neurobehavioral deficits in the absence that below a blood lead level of 80 /ig/dl some of any classical signs of lead encephalopathy. The behavioral changes did occur in adult workers. In literature on this subject is somewhat limited and addition, variability of performance increased with controversial but still allows for certain statements increasing blood lead level; however, only during to be made about the possible hazard of low to period of high-demand performance did a worker's moderate lead exposure levels. capacity clearly decrease as a result of lead ex­ If such neurobehavioral deficits occurred in posure. Unfortunately, aspects of the Morgan and adults with great frequency, one might expect this to Repko work can be criticized because of be reflected by higher rates of absences or reports of methodological problems, including reported ap-

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paratus failures during testing of subjects. Also, actually responsible for producing the neuropathies, findings analogous to those reported by Morgan and it appears that in at least one case220 blood levels al­ Repko were not obtained in a similar study215 that ways below 70 /ig/dl were sufficient to cause found no differences between control and lead-ex­ peripheral nerve dysfunctions. That study by posed workers on a number of psychometric and Seppalainen220 was also generally methodologically other performance tests. sound, having been well controlled for the possible In addition to the above study213 suggesting possi­ effects of extraneous factors such as temperature ble CNS dysfunctions, numerous investigations have differences at the nerve conduction velocity assess­ provided electrophysiological data indicating that ment sites. On the other hand, it should be noted that peripheral neuropathy symptoms are associated at the data reported for control subjects were obtained times with blood lead values < 80 /ig/dl. As at an earlier time (1971 to 1973) than data for the reviewed by Seppalainen,216 reductions in nerve lead exposed subjects (early 1973); and no blood conduction velocities and electromyographic lead levels were reported for the control subjects. deficits have been observed in patients with known Still, when the Seppalainen220 results are viewed col­ lead poisoning but without clinical neurological lectively with the data from other studies reviewed symptoms.217219 More recently, such peripheral above, strong evidence appears to exist for periph­ nerve deficits were established by Seppalainen220 for eral neuropathies occurring in adults at blood lead lead workers whose blood lead levels were as low as levels of 50 to 70 /ig/dl or, possibly, at even lower 50 /ig/dl and had never exceeded 70 /ig/dl during levels. their entire exposure period (mean = 4.6 years), as In addition to suspected neurobehavioral effects determined by regular monitoring. Similar results of relatively low-level lead exposures in adults, 221 were obtained in a study by Melgaard et al. on au­ there is an increasing concern that low-level ex­ tomobile mechanics exposed to TEL and other lead posures producing blood lead levels of 40 to 80 compounds in lubricating and high-pressure oils. /xg/dl (or even less) in children may induce subtle Results ofa multielemental analysis ofthe worker's neurological damage, especially to the very young blood for lead, chromium, copper, nickel, and developing CNS. This issue has attracted much at­ manganese indicated a clear association between tention and generated considerable controversy dur­ lead exposure and peripheral nerve damage. Half of ing the past decade. The evidence for and against the the workers (10 of 20) had elevated blood lead occurrence of significant neurobehavioral deficits at levels (60 to 120 /ig/dl) and showed definite relatively low levels of lead exposure is, at this time, electromyographic deficits. Mean blood lead level quite mixed and largely interpretable only after a for the control group was 18.6 /ig/dl. Melgaard et thorough critical review of the methodologies al.221 reported additional results on associating lead employed in each ofthe various important studies on exposures with polyneuropathy of unknown etiology the subject. in 10 cases from the general population. Another One of the major approaches that has been study reported recently by Araki et al.222 provides employed is the retrospective analysis of lead levels further confirmation of the Seppalainen220 and existing in populations of apparently asymptomatic Melgaard et al.221 findings in that evidence for pe­ children that are then divided into nonexposed con­ ripheral neuropathy effects were reported for lead- trol and one or more lead-exposed experimental industry workers with blood lead values of 29 to 70 groups for comparisons of their performance in /ig/dl. The very low blood lead levels, below 50 various neurological and psychometric tests. A few /ig/dl, reported in some of the above studies, studies have been followed by subsequent further however, should probably be viewed with caution reevaluation of the same children by the same in­ until further confirmatory data are reported on sam­ vestigators in an effort to assess whether indications ples of larger size using well verified blood assay of continuing neurobehavioral impairment still ex­ results. isted. Among the major studies that have employed In summary, the above studies, when taken this basic approach and that are widely cited in together, appear to provide reasonably strong evi­ regard to this issue, those of de la Burde and dence that subclinical peripheral neuropathies occur Choate,223224 Perino and Ernhart,225 Albert et in some adults having blood lead levels in the 50 to al.,226 and Landrigan et al.227 suggest significant 70 /ig/dl range. Furthermore, although it could be effects of asymptomatic, low-level lead exposure. In argued that substantially higher lead body burdens contrast, the studies of Kotok,228 Lansdown et al.,229 existing before the time of some of the studies were and McNeil et al.230 report generally negative

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results. Two other studies, one by Landrigan et al.231 ate use of multiple chi-square statistical analyses in and one by Kotok et al.,232 although not reporting the manner employed to analyze the results of the clearly statistically significant differences between study. Upon recomputation ofthe statistical signifi­ moderately lead-exposed and control subjects, cance of observed differences, by means ofthe more nevertheless report certain findings that are highly appropriate Fisher's exact probability test and ac­ suggestive of a relationship between moderate lead counting for the number of tests conducted, several exposure and cognitive impairment. measures originally reported to be statistically sig­ Among the several studies presenting evidence for nificant still turn out to be significant at p <0.05 or CNS deficits being associated with blood levels of lower. One last problem relates to ambiguities in less than 80 figldl are the work of de la Burde et subject selection that complicate interpretation of 225 al 223,224 an(- perino and Ernhart. De la Burde et the full meaning ofthe results obtained. Because it is al.223 observed dysfunctions ofthe CNS, fine motor stated that the lead-exposed group included dysfunction, impaired concept formation, and children with blood lead levels of 40 to 100 /ig/dl, altered behavioral profile in 70 preschool children or of at least 30 /ig/dl with "positive radiographic exhibiting pica and elevated blood lead levels (in all findings of lead lines in the long bones, metallic cases above 30 /ig/dl, mean = 59 /ig/dl) in com­ deposits in the intestines, or both," the reported parison to matched control subjects not engaging in deficits might be readily attributed to blood lead pica. In a follow-up study on the same children (at 7 levels as low as 30 /ig/dl. Other evidence,101 to 8 years old), de la Burde224 reported further however, suggests that such a simple interpretation confirmation of continuing CNS impairment as may not be completely accurate. That is, the work of assessed by a variety of psychological and neurologi­ Betts etal.101 indicates that lead lines are usually not cal tests. This was despite the fact that many of the seen unless blood levels exceed 60 /ig/dl for most blood lead levels of the lead-exposed children had children at some time during exposure, although by then dropped significantly from the initial study. some (approximately 25 percent) may show lead In general, the de la Burde et al.223224 studies appear lines at blood lead levels of 40 to 60 /ig/dl. Vir­ to be methodologically sound, having many features tually none have lead lines at levels below 40 /ig/dl. that strengthen the case for the validity oftheir find­ In view of this, the de la Burde results probably can ings. For example, there were appreciable numbers be most reasonably interpreted as demonstrating of children (67 lead-exposed and 70 controls) whose lasting neurobehavioral deficits at blood lead levels blood lead values were obtained in preschool years in excess of 50 to 60 /ig/dl. and who were old enough (7 years) during the Similar conclusions are also warranted on the follow-up study to cooperate adequately for reliable basis of results of the Perino and Ernhart study,225 psychological testing. The specific psychometric which demonstrated a relationship between tests employed were well standardized and accepted neurobehavioral deficits and blood lead levels rang­ as sensitive indicators of minimal brain damage, and ing from 40 to 70 /ig/dl in a group of 80 inner-city the neurobehavioral evaluations were carried out preschool black children. One of the more interest­ blind, that is, without the evaluators knowing which ing aspects ofthe findings, is that the normal correla­ were control or lead-exposed subjects. tion of .50 between parent's intelligence and that of The de la Burde223-224 studies might be criticized their offspring was found to be reduced to only . 10 in on several points, none of which in the final analysis the lead-exposed group, presumably because of the provide sufficient grounds for rejecting their influence of another factor (lead) that interfered validity. One difficulty is that blood lead values with the normal intellectual development of the were not determined for control subjects in the in­ lead-exposed children. Many ofthe methodological itial study, but the lack of history of pica, as well as virtues of the de la Burde studies223224 were also tooth lead analyses done later for the follow-up present in the Perino and Ernhart225 work, and study, render it very improbable that appreciable blood lead determinations and statistical analyses numbers of lead-exposed subjects might have been appeared sound. About the only alternative ex­ wrongly assigned to the control group. Also, results planation for these results might be differences in the indicating no measurable coproporphyrins in the educational backgrounds of the parents of the con­ urine of control subjects at the time of initial testing trol subjects when compared with lead-exposed sub­ further help to confirm proper assignment of those jects, because parental education level was found to children to the nonexposed control group. A second be significantly negatively related to the blood lead point of criticism addresses the probably inappropri­ levels of the children participating in this study.

11-20 Parents of children in the lead-exposed group had McCarthy Subscales are plotted graphically, as in significantly poorer educational backgrounds than Figures 11-2 and 11-3, then a rather interesting the control group parents. The importance of this relationship between test performance and levels point lies in the fact that several other studies233235 arid duration of lead exposure becomes apparent. have demonstrated that the higher the parental 1 1 1 1 education level, the more rapid the development 95 and the higher the intelligence quotients (I.Q.'s) of their children. It is nevertheless interesting that the de la Burde studies and the Perino and Ernhart work 90 V - point to essentially the same conclusion, i.e., that neurobehavioral deficits occur at blood lead levels 85 \ - possibly as low as 40 /ig/dl. Also, in both cases, the children studied were from inner-city, low-income areas. 80 \ - Two other studies with positive findings had, for 75 * — the most part, some serious methodological limita­ tions. Albert et al.226 found that asymptomatic ki 1 1 children (5 to 15 years old) whose blood lead levels CONTROLS SHORT-TERM LONG-TERM ENCEPHALOPATHY

at an earlier age were elevated (> 60 /ig/dl) later DEGREE OF LEAD EXPOSURE had significantly more mental disorders and poorer Figure 11-2. McCarthy General Cognitive Index scores as a school performance than a control group with lower function of degree of lead exposure.211 lead levels in both blood and deciduous teeth. Un­ fortunately, however, no assay ofthe lead burden, in T T either blood or teeth, was done for about one-half of the children in the control group; and no significant effects were reported for children with lead levels < 60 /ig/dl. Also, another major criticism is that some children in the control group had relatively high blood lead levels (> 40 /ig/dl). In another study, Landrigan et al.227 found that asymptomatic, lead- exposed children living near a smelter scored signifi­ cantly lower than matched controls on measures of MOTOR PERCEPTUAL performance I.Q. and finger-wrist tapping. The con­ VERBAL trol children in this study were, however, not well MEMORY matched by age or sex to the lead-exposed group, QUANTITATIVE although it should be pointed out that results re­ mained statistically significant even after appropri­ CONTROLS SHORT-TERM LONG-TERM ENCEPHALOPATHY

ate adjustments were made for age differences. DEGREE OF LEAD EXPOSURE In another relevant study, presented in a doctoral Figure 11-3. Scores on McCarthy Subscales as a function of dissertation by Rummo,211 significant degree of lead exposure.211 neurobehavioral deficits were found (hyperactivity, Although the scores for short-term moderate-ex­ lower scores on McCarthy scales of cognitive func­ posure subjects are essentially the same as control tion, etc.) for children who had previously ex­ values, an interesting aspect is that the scores for perienced high levels of lead exposure that had pro­ long-term moderate-exposure subjects consistently duced acute lead encephalopathy. Mean maximum fall below those for control subjects and lie between blood lead levels for those children at the time of en­ the latter and the encephalopathy group scores. cephalopathy were 88 ± S.D. 40 /ig/dl. Children Thus, it would appear that long-term moderate lead with moderate degrees of blood lead elevation, exposure may, in fact, exert subtle neurobehavioral however, were not significantly different from con­ effects. This might be shown to be statistically sig­ trols on any measure of cognitive functioning, psy­ nificant by means of other types of analyses or if chomotor performance, or hyperactivity. On the larger samples were assessed. It should be noted that other hand, if the data for performance on the (1) the maximum blood lead levels for the short- McCarthy General Cognitive Index or several term and long-term exposure subjects were all >40

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/ig/dl (means = 61 ± S.D. 7 and 68 ± S.D. 13 TABLE 11-3. COMPARISON OF TEST RESULTS IN LEAD 232 /ig/dl, respectively), whereas control subjects all AND CONTROL GROUPS had blood lead levels below 40 /ig/dl (mean = 23 ± Ability class Group Mean I.Q. ± S.D. Significance S.D. 8 /ig/dl); and (2) the control and lead-exposed subjects were inner-city (Providence, Rhode Island) Social maturity Lead 123.5 ± 22.7 p>.10 Control 126.3 ± 17.7 children well matched for socioeconomic back­ ground, parental education levels, incidence of pica, Spatial Lead 92.0 ± 18.0 .10 >p >.05 and other pertinent factors. Control 100.8 ± 18.7 A somewhat similar pattern of results emerged Spoken vocabulary Lead 91.7 ± 13.9 p>.10 from a more recent study by Kotok et al.232 in which Control 92.9 ± 13.7 36 Rochester, New York, control group children Information- Lead 94.5 ± 14.9 p>.10 with blood lead levels < 40 /ig/dl were compared comprehension Control 96.3 ± 16.6 with 31 asymptomatic children having distinctly ele­ Visual attention Lead 89.7 ± 18.3 p>.10 vated blood lead levels (61 to 200 /ig/dl). Both Control 93.3 ± 23.0 groups were well matched on important background Auditory memory Lead 93.0 ± 24.0 p >.10 factors, including, notably, their propensity to ex­ Control 99.9 + 32.0 hibit pica. Again, no clearly statistically significant (p <.05) differences between the two groups were using various standardized psychometric techniques, found on a number of different tests of cognitive and neurologic examinations, and ratings by teachers, sensory functions. parents, or experimenters. For example, Kotok228 As indicated, however, by test results from the reported earlier that developmental deficiencies Kotok et al. study232 presented in Table 11-3, the (using the Denver Development Screening test, mean scores ofthe control-group children were con­ which is a somewhat insensitive measure of develop­ sistently higher than those ofthe lead-exposed group ment) in a group of asymptomatic children having for all six of the ability classes listed. Also, in one elevated lead levels (58 to 137 /ig/dl) were identical case the level of significance achieved borderline to those in a control group similar in age, sex, race, significance (.10 >p >.05), a pattern of results that environment, neonatal condition, and presence of hints at a trend existing toward lower ability levels pica, but whose blood lead levels were lower (20 to for the lead group. The authors cautiously stated 55 figldl). The deficiencies could be correlated with that "the data do not prove that these children have inadequacies in the children's environment. sustained no neurologic damage by lead" and that Children in the lead-exposed group, however, had "later longitudinal testing may demonstrate cogni­ blood lead levels as high as 137 /ig/dl, whereas some tive or educational deficiencies." They also indi­ of the controls had blood lead levels as high as 55 cated that "evaluation of behavior, neurologic, or /ig/dl. Thus, the study was in effect a comparison of motor functioning was not carried out" and noted two groups with different degrees of elevation in that "subtle cognitive and fine motor changes were lead exposure rather than one of lead-exposed ver­ demonstrated in an extensive and carefully con­ sus nonexposed control children. trolled evaluation of asymptomatic children resid­ In several studies of children living in the vicinity ing in the vicinity of an El Paso lead smelter."227 of smelters or factories, significant neurobehavioral They go on to imply that the earlier exposure to lead effects have typically not been found at moderate in infancy of the El Paso children and their longer elevations of blood lead levels. For example, period of exposure (mean = 6.6 years versus < 3 Lansdown et al.229 found a relationship between years for the Rochester group) might account for the blood lead level in children and the distance they disparity in results .between the two studies. Their lived from lead-processing facilities, but no rela­ results, on the other hand, plus the pattern seen in tionship between blood lead level and mental func­ the Rummo2" study, can be construed as evidence tioning was found. Only a minority of the lead-ex­ consistent with the findings of the de la Burde posed sample had blood lead levels over 40 /ig/dl, studies223224 and others reviewed above that report however, one would not expect a striking relation­ results linking low to moderate levels of lead ex­ ship between mental functioning and lead levels be­ posure to significant behavioral impairments. low such a level. Other studies have produced mixed or negative In an extensive, generally thorough study, McNeil results in attempts to determine whether a relation­ et al.230 found that a sample of children living near a ship exists between lead exposure and CNS deficits lead smelter in El Paso was comparable,medically

11-22 and psychologically to matched controls living utilized in other studies in an effort to demonstrate elsewhere in the same city other than in the direct that low or moderate blood lead levels cause signifi­ effects of lead (blood lead level, free erythrocyte cant neurobehavioral deficits. This approach con­ protoporphyrin levels, and X-ray findings). Lead- sists of identifying populations of children with diag­ exposed children in the group living near the smelter nosed neurobehavioral deficits of unknown etiology did, however, have significantly different per­ and assaying blood lead or making other assessments sonality test results, which were ascribed by the in order to link past lead exposures to the children's authors as being due to recent upheaval in the lives present neurobehavioral impairments. Thus, for ex­ of the lead-exposed children who had been recently ample, efforts have been made to implicate moder­ forced to move from the vicinity ofthe smelter. Con­ ate or low level lead exposures as a causative factor siderable community unrest existed at the time of in at least some cases of hyperactivity of unknown both the McNeil et al. study230 and the work of etiology. The possibility that such low-level lead ex­ Landrigan et al.227 on the El Paso smelter area posures induce hyperactivity has gained credence population, which grew out of circumstances associ­ through the well documented87203 fact that hyperac­ ated with the discovery of the lead exposures and tivity is one of the frequent neurobehavioral se­ disposition of legal matters surrounding them. The quelae observed in children who survive episodes of impact of the extraneous unrest on both studies has acute encephalopathy resulting from high-level lead tended to cloud interpretation of the true meaning of exposures. The evidence for and against the hy­ their respective results which in turn have become pothesis that low-level lead exposures produce hy­ quite controversial. See Appendix E for more infor­ peractivity has been accumulating at a rapid rate mation. The personality test results ofthe McNeil et during the past few years and has generated con­ al.230 study could nevertheless have been caused by siderable controversy on the subject. Only a few of the effects of lead on the exposed group. Also, it the more salient findings are viewed below and in should be noted that a few suggestive trends toward the section on animal studies (Section 11.5.2). statistical significance for certain interactions be­ In a case-control study, David et al.236 compared tween lead and age of subjects (p <.10) were re­ the incidence of elevated blood lead levels in five ported for some of the cognitive-function test groups of children: (1) a pure hyperactive group measures. with no apparent cause for hyperactivity; (2) a group Based on the above results, the authors of the of hyperactive children with a highly probable cause Lansdown et al.229 and the McNeil230 papers con­ of hyperactivity, e.g., prematurity; (3) a group of hy­ cluded that no evidence was found for the occur­ peractive children with a possible cause; (4) a group rence of neurobehavioral effects at subclinical lead of children who had recovered from lead poisoning; exposure levels in their studies. Perhaps that conclu­ and (5) a nonhyperactive control group. Pure hy­ sion could be generalized to suggest that no signifi­ peractive children had statistically significantly cant CNS deficits typically occur as a result of higher blood lead levels (mean = 26.2 ± 8 figldl) subclinical, low to moderate lead exposures of than controls (mean = 22.2 ± 9.6 /ig/dl), whereas children living in the vicinity of smelters or other children with a highly probable cause did not (mean lead-processing facilities. To the extent that those = 22.9 ± 6.6 /ig/dl). Similarly, the pure hyperactive children may differ in significant ways from the children tended to excrete more lead than controls inner-city children shown by de la Burde,223-224 or probable cause hyperactives when given a single Perino and Ernhart,225 and Albert226 to have d©se of penicillamine. Although the causal relation­ neurobehavioral impairments at blood lead levels as ship between lead exposure and hyperactivity cannot low as 40 to 50 figldl, ambiguous results from the be said to be proved by this study, the data of David smelter children are not necessarily contradictory to et al.,236 when placed with findings of hyperactivity the better established findings of significant CNS in children known to have recovered from lead effects for inner-city children. Furthermore, it poisoning and numerous animal studies demonstrat­ should be noted that reports of peripheral neuropa­ ing alterations in motor activity following lead ad­ thies for both populations of children at low to ministrations, might be interpreted as supporting the moderate lead exposure levels, as described, may in­ hypothesis that a relationship between moderate dicate that both groups are at significant risk for at lead exposure and altered motor activity exists. least that type of neural tissue damage. On the other hand, several other points argue An additional approach, different from the basic against acceptance of such a thesis at this time. For strategy employed in the above studies, has been one thing, the David et al.236 study itself and the

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author's conclusion6 can be questioned on several its predictive value was well served by cobalt and bases. In that study, for example, the closeness ofthe cadmium because of a significant negative correla­ match of subjects in the five groups oij variables tion between lead and cobalt (r = .67;p <.01) and a other than age and sex is not clearly specified by the significant positive correlation between lead and authors. Also, the interpretation of differences in cadmium (r = + .53; p <.01). Unfortunately for blood lead levels in the 7-to-8-year-old children is present purposes, no blood lead levels or possible fraught with numerous problems, not #ie least of past exposure histories were provided for the which is the fact that such levels are probably not children in the above study.240 very accurate indices of long-past lead exposures Other recent studies analogous in basic approach that presumably occurred during presclhool years. to that employed by David et al.236 and Pihl and Many factors in the interim between presumed lead Parkes240 have provided intriguing new information exposure and assay for lead could affect the results, tending to link prenatal lead exposures to the later including possible differentially higher incidences of development of mental retardation. For example, pica in the hyperactive children than in control sub­ Beattie et al.241 identified 77 retarded children and 237 jects. Klein et al. have noted that pica may be part 77 normal children matched on age, sex, and of certain behavioral syndromes that exist even in geography. The residence during the gestation ofthe the absence of lead exposure, but that would pre­ subject was identified, and a first-flush morning dispose the affected child toward more lead inges­ sample oft?;** water was obtained from the residence. tion by virtue ofthe habit's presence. Indteed, there is Of 64 matched pairs, no normal children were found evidence that among mentally subnormal children to come from homes served with water containing whose mental deficiency can be definitely attributed high lead levels (>800 /ig/liter), whereas 11 of the to etiologies other than lead poisoning that there is 64 retarded children came from homes served with both a high incidence of pica and modterately ele­ water containing high lead levels. The authors con­ 238 vated blood lead. Last, it should be noted that a clude that pregnancy in a home with high lead in the 2 number of other investigators, ".227,229.230,239 wno water supply increases by a factor of 1.7 the risk of expressly looked for evidence of lead-induced hy­ bearing a retarded child. peractivity as part of their screening for 242 neurobehavioral deficits associated with blood lead In a follow-up to the Beattie study, Moore et al. levels as low as 40 figldl, failed to find any signifi­ obtained lead values from blood samples drawn cant effects that support the thesis that low-level during the second week of life and stored on filter lead exposures induce hyperactivity. Thus, even paper. These samples had been obtained as part of a though the hypothesis is intriguing and certainly routine phenylketonuria screening study and were worthy of further investigation, it cannot be stated at kept on file. Blood samples were available for 41 of this time that sufficient evidence exists to establish the retarded and 36 of the normal children in the hyperactivity as a neurobehavioral deficit clearly as­ original study by Beattie. Blood lead concentrations sociated with low or moderate lead exposures. in the retarded children were significantly higher than values measured in. normal children. Mean In addition to the above data bearing on possible blood lead for retardates was 1.23 ± 0.43 links between subclinical lead exposures and the in­ /iMol/liter (25.5 ± 8.9 /ig/dl) and for normals was duction of hyperactivity, certain recently reported 1.0 ± 0.38 /iMol/liter (20.9 ± 7.9 figldl). The data240 provide evidence implicating increased difterence in lead concentrations were significant heavy metal absorption, including lead uptake, in (p = 0.0189) by the Mann-Whitney test. the etiology of learning disabilities. More These two studies suggest that lead exposure to the specifically, children identified for other classifica­ fetus during the critical period of brain development tion purposes as having learning disabilities were may cause perturbations in brain organization that found to have significantly elevated levels of lead, as are expressed later in mental retardation syndromes, well as cadmium and some other metals,, in their hair and they raise for careful scrutiny the risks of in­ when compared with control children not classed as trauterine exposure to lead. Insufficient information learning disabled. In fact, a discrimin»ant function exists, however, to allow estimation ofthe levels of analysis yielded 98 percent accuracy im classifying lead exposure of pregnant women that might cause children as normal or learning disabled based on a those prenatal effects in the fetus that may result in combined factor of cadmium, cobalt, manganese, later neurobehavioral impairments. chromium, and lithium levels. Lead was not in­ One last adverse effect of lead on neural function cluded in this five-metal discriminant function, since in children remains to be considered, and that is the

11-24 possible induction of peripheral neuropathies by be made regarding any threshold for a pathologic low-to-moderate lead exposures. It is generally ac­ slowing of NCV. cepted that lead-induced peripheral neuropathies, llll llll although frequently seen in adults after prolonged YICONDUCTION VELOCITYI = 54.8 - .045 x (BLOOD LEAD) exposures, are extremely rare in children. Several _ Ir •= • 0.381 In = 202) articles243"245 in the literature, however, do describe " case histories that confirm the occurrence of lead-in­ — • duced peripheral neuropathies, as indexed by • electromyography, assessments of nerve conduction • velocity, and observations of other overt neurologi­ — • cal signs, such as tremor, wrist and foot drop, etc. • • > 62.00 Some of these frank neuropathic effects have been z O _••••• • • observed for several cases at blood lead levels of 60 o 56.80 —- •• . •V# • amIa • • «••..•• •"#• •• • **. - to 80 /ig/dl,245 and in other cases peripheral —*!%*&*+9 ! : neuropathy was associated with blood lead values of ° 51.60 - • mzTr —*"y -»-jL • 30 /ig/dl; however, in the latter cases, lead lines in • • a a a a • • — long bones suggest probable past exposures leading . • * • • to prior blood lead levels at least as high as 40 to 60 • • /ig/dl and probably in excess of 60 /ig/dl (based on . • • 101 1 1 1* 1 1 1 1 1 the data of Betts et al. ). In each ofthe present case 0 15 30 45 60 75 90 105 120 135 150 studies, there was reported some, if not complete, BLOOD LEAD,

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It should be emphasized that once encephalopathy were responsible for the behavioral deficits ob­ occurs death is not at all an improbable outcome, served. Monitoring of lead exposures in the subjects regardless of the quality of medical treatment has in all cases been highly intermittent during the available at the time of any acute crisis. In fact, cer­ period of life preceding the behavioral assessment. tain diagnostic or treatment procedures themselves In most cases, only one or two blood lead values are tend to exacerbate matters and push the outcome provided per subject. toward fatality ifthe nature and severity ofthe prob­ 11.5.2 Animal Studies lem are not fully recognized or are misdiagnosed. It 52 is also crucial to note the rapidity with which acute Pentschew and Garro initially described an encephalopathy symptoms or death can develop in animal model of lead encephalopathy in which apparently asymptomatic individuals or in those morphological changes occurred similar to those re­ only apparently mildly affected by elevated body ported in children. Neonatal rats were exposed to burdens of lead. It is not unusual for rapid lead by feeding mothers a diet containing 4 percent deterioration to occur, with convulsions or coma lead carbonate. The lead was then transmitted to the suddenly appearing and progressing to death within suckling young via the mothers' milk. Between 23 48 hr. This strongly suggests that even in apparently and 29 days of age, 90 percent of the animals asymptomatic individuals rather severe neural developed paraplegia lasting no longer than 2 damage probably does exist at high blood lead levels weeks. Eighty-five to 90 percent of the paraplegic even though it is not yet overtly manifested in ob­ animals died during this period. vious encephalopathic symptoms. This tends to be Neuropathological examination of these animals borne out by studies showing that children having revealed capillary activation, glial proliferation, high blood lead levels (over 80 to 100 /ig/dl), but areas of transudation, and spotty hemorrhages, pri­ not observed to manifest acute encephalopathy marily in the cerebellum and the striatum. The white symptoms, are permanently, cognitively impaired, matter of the cerebral hemispheres, especially the as are individuals who survive acute episodes of lead corpus collosum, was also involved, though to a encephalopathy. lesser extent. Ischemic neuronal changes in focal Other evidence tends to confirm that some type of distribution were rare and were localized in the 52 neural damage does exist in asymptomatic children, cerebral cortex. Pentschew and Garro concluded and not necessarily only at very high levels of blood that the lead encephalopathy ofthe suckling rat was lead. The body of studies on low- or moderate-level caused by a disorder in the permeability of the lead effects on neurobehavioral functions, as sum­ capillaries, resulting in dysoric encephalopathy. The marized in Table 11-4, present overall a rather im­ suckling rat therefore differs from the human in that pressive array of data pointing to that conclusion. the latter shows a mixture of both dysoric and Several well-controlled studies have found effects hemodynamic alterations. Further verification of the dysoric nature of lead encephalopathy in the that are clearly statistically significant, whereas 53 others have found nonsignificant but borderline suckling rat was provided by Lampert et al., who effects. Even some studies reporting generally non­ used either colloidal thorium dioxide (Thorotrast) or Trypan Blue, neither of which normally pene­ significant findings at times contain data confirming trates the blood-brain barrier. In suckling rats statistically significant effects, which the authors at­ poisoned with 4 percent lead carbonate, however, tribute to various extraneous factors. Another way both Thorotrast and Trypan Blue were found to to look at the situation is to consider that elevated penetrate the striatum and cerebellum. blood lead level is the single common factor extant in all of the groups showing significant behavioral Since the initial description by Pentschew and deficits in the different studies. It should also be Garro,52 lead encephalopathy in the rat has been noted that, given the'likely subtle nature of some of replicated by Thomas et al.246 Michaelson and the behavioral or neural effects probable at low Sauerhoff,247 and Krigman and Hogan.248 levels of lead exposure, one would not expect to find 249 striking differences in every instance. The blood Clasen et al. reported lead encephalopathy in lead levels associated with neurobehavioral deficits juvenile Rhesus monkeys exposed to 0.5 g of lead in asymptomatic children appear to be in excess of per day for 6 to 18 weeks. They reported 50 to 60 /ig/dl. Great uncertainties remain, morphological changes similar to those occurring in however, as to whether these exposure levels (ob­ humans, which consisted of edematous changes in served blood lead levels) represent the levels that the cerebellar and subcortical white matter. Diffuse

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TT'TTT- '-- -"

TABLE 11-4. SUMMARY OF RESULTS OF HUMAN STUDIES ON NEUROBEHAVIORAL EFFECTS AT MODERATE BLOOD LEAD LEVELS

Population Age at Blood lead. Psychometric Summary of results Levels of Reference studied N/group testing, yr mm tests employed (C= control; Pb= lead)3 significance" De la Burde and Inner city Control = 72 4 Not assayed' Stanford Binet IQ C= 94 Pb= 89 p <.05 Choate (1972)2*3 (Richmond, VA) Lead= 70 4 40-100" Other measures C >Pb on 3/4 tests N.S.-P <.01 Oe la Burde and Followup Control =67 7 See above" WISC Full Scale IQ C=90Pb=87 p <.01 Choate (1974)22* same subjects Lead= 70 7 See above" Neurologic exam C better than Pb p<.01 Other measures C >Pbon9/10 N.S.p <.001 tests Perino and Inner city Control =50 3-6 10-30 McCarthy General Ernhart (1974)225 (New York, NY) Cognitive C= 90 Pb= 80 p <.01 Lead= 30 3-6 40-70 McCarthy Subscales OPbon 5/5 scales N.S.p <.01 Pueschel etal. Inner city Control = 56 ? <40 Stanford-Binet IQ C= ?Pb= 86 — (1972)99 (Boston, MA) Lead= 56 ? >40 Neurologic exam C better than Pb p<.001 Landrigan et al. Smelter area Control =46 3-15 (x= 9.3) <40 WISC Full Scale IQ< C=93Pb=87 N.S. (1975)22? (El Paso, TX) Lead= 78 3-15 (x= 8.3) 40-68 WPPSI Full Scale IQ- C= 91 Pb= 86 N.S. WISC + WPPSI Combined C= 93 Pb= 88 p <.01 WISC + WPPI OPbon Subscales 13/14 scales N.S.p <,01 Neurologic testing C >Pb on 4/4 tests N.S.p <.001 Rummo (1974)2" Inner city Control Ss= 45 4-8 (5=5.8) x= 23± 8 McCarthy General C= 93; S= 94; N.S.p <,01 (Providence, Rl) Cognitive L= 88; P= 77 (pvsC) Short Pb Ss= 15 4-8 (x**= 5.6) 7= 61± 7 McCarthy Subscales C+S>L>Pon5/5 ' tests N.S.p <.01 . (pvsC) Long Pb Ss= 20 4-8 (x = 5.6) x= 68± 13 Neurologic exam C+SL>P rating on ratings N.S.

Post enceph Pb= 10 4-8 (x= 5.3) x= 88 ± 40 Objective neuro­ C+S>L>Pon N.S.p <.01 logic tests 3/12 tests (pvsC) Kotok et al. Inner city Control=36 1.9-5.6 (x= 3.6) 11-40 IQEfor six ability IQ Equivalent for (1977)232 (Rochester, NY) classes: each: Lead=31 1.7-5.4 (x= 3.6) 61-200 Social maturity; C= 126Pb=124 p <.10for spatial Spatial relations; C= 101 Pb= 92; Spoken vocab; Info, C= 93 Pb= 92; p >. 10for all comprehension; C= 96 Pb= 95; other ability Visual attention; C= 93 Pb= 90 classes Auditory memory C= 100 Pb= 93 Kotok (1972)228 Inner city Control = 25 1.1-5.5 (x= 2.7) 20-55 Denver OPbon 1/3 N.S. (New Haven, CT) Lead= 24 1.0-5.8 (x= 2.8) 58-137 Developmental Subscales Scale Lansdown et al. Inner city Control = 172 616 <40 WISC C= 100 N.S. (1974)229 (London, England) Lead= 43 616 40-60+ WAIS Pb= 101105 McNeil etal. Smelter area Control= 61-152 1.5-1.8(Mdn= 9) McCarthy General <40 (1975)230 (El Paso, TX) Cognitive C=82Pb=81 N.S. Lead= 23-161 1.5-1.8(Mdn= 9) WISCWAIS Full >40 Scale IQ C=89Pb=87 N.S. Oseretsky Motor Level C= 101 Pb= 97 N.S. California Person­ ality C= 80 Pb= 72 N.S. Frostig Perceptual Quotient C= 100 Pb= 103 N.S. Finger-Thumb Apposition C= 27 Pb= 29 N.S.

a Mean test scores obtained for control children are indicated by C = T; mean scores for respective lead-exposed groups are indicated by Pb = x (except for Rummo'--' study where C = control, S = short-term lead-ex­ posed subjects; L = long term lead-exposed group and P = postencephalopathy lead group). bN.S. = nonsignificant, i.e. p >.05. Note exception of p <.05. Note exception of p <. 10 listed for spatial ability results in Kotok et al.232 study. ^Urinary coproporphyrin levels were not elated. <- Or 30 /xg/dl or above with positive radiologic findings. The latter suggest earlier exposure in excess of 50-60 Mg/dl. e Assays for lead in teeth showed the Pb-exposed group to be approximately twice as high as controls (202 /ig/g vs. 112 /ig/g, respectively). Used for children over 5 years of age. 8 Used for children under 5 years of age.

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astrocytosis and glial nodules were found in white neuron cannot be definitively concluded from these matter along with perivascular exudate. studies. Lead encephalopathy in the mouse was first de­ The relative involvement of the capillary bed in scribed by Rosenblum and Johnson250 who, like the lead encephalopathy may depend on the degree Pentschew and Garro, used the suckling animal. In of maturation at the time of exposure. Bouldin et contrast to the rat brain, the striatum and al.252 for example, were able to produce lead en­ cerebellum of mice fed either 0.5 or 1 percent lead cephalopathy in adult guinea pigs with no cerebral carbonate displayed only faint staining with Trypan edema or increased capillary permeability, whereas Blue (except for a single animal with darkly stained the suckling rat shows these effects almost ex­ cerebellum) and only occasional paraplegia. The clusively. most striking vascular change in the poisoned mice Since the initial description of lead encephalopa­ was the appearance of intervascular strands thy in the rat by Pentschew and Garro,52 considera­ throughout the brain, especially in the hippocampus ble effort has been made to define more closely the 250 and basal ganglia. Rosenblum and Johnson con­ extent of CNS involvement at subencephalopathic cluded that no cerebral edema and focal destructive levels of lead exposure. This experimental effort has lesions were found in the mouse brains. Therefore, focused almost exclusively on the developing organ­ the histological response of the suckling mouse ex­ ism. The interpretation ofa large number of experU posed to lead differs from that seen in the suckling ments dealing with early exposure to lead have, rat. These differences are probably not attributable however, been confounded by a number of flaws in to different exposure levels (4 percent carbonate in experimental design. the rat versus 1 percent in the mouse), because the Perhaps the most notable of these experimental food consumption per unit body weight is three to shortcomings has been the presence of undernutri­ four times greater in the mouse than in the rat, mak­ tion in experimental animals. Changes in nutritional ing the external exposures roughly equivalent. status during early brain development are known to Wells et al.251 have recently reported on experi­ produce changes in behavior.254255 Castellano and mental lead encephalopathy in calves. Four Jersey Oliverio,256 for example, reported a marked delay in bull calves were exposed to 20 mg/kg/day of lead neurological development, an increase in explorato­ acetate, beginning at 3 months of age and lasting for ry locomotor activity, and a lowered avoidance per­ 8 to 273 days. Histological evaluation ofthe brains formance in mice that were undernourished during of exposed animals revealed focal vacuolation ofthe early development by being reared in large litters. neuropil, neuronal necrosis, and changes in the Neurochemical processes have also been shown to be capillary walls in the cerebral cortex and in subcor­ affected by early undernutrition. Eckhert et al.257 re­ tical areas. Lesions in these cattle were similar to ported changes in cholinergic enzyme activities those seen in children with lead encephalopathy in when rats were placed on protein-deficient diets that both show not only vascular changes in the during various periods of development. Their brain with edema but also areas of neuronal results indicate that "the relationship between the necrosis. activity of individual cholinergic enzymes, nutri­ In summary, the histopathological changes as­ tional status and developmental age is complex and sociated with lead encephalopathy vary among is not the same for different brain regions or even the species and are characterized by their relative in­ same brain region exposed to undernutrition during volvement of neuronal degeneration and vasculopa­ different periods of development." Therefore, in thy. The fact that lead encephalopathy is reported to reviewing the animal literature concerned with the occur in the absence of cerebral edema in man,198 neurotoxicology of lead exposure, the possible con­ mouse,250 and guinea pig252 is argument for a direct tribution of undernutrition must be considered. neuronal involvement of lead. Recent studies have, Furthermore, the possibility also exists that lead and in fact, suggested direct neuronal alterations by undernutrition may be having a synergistic effect on lead. Bull et al.253 reported that lead interferes nervous system development. In this case, the with potassium-stimulated respiration of rat methods of pair-feeding currently employed in some 247 258 cerebral cortex slices, and Nathanson et al.17 re­ studies - may not provide adequate control for ported that lead inhibited brain adenyl cyclase in this undernutrition. Examples of dietary factors vitro. No distinction can be made in these studies, known to affect susceptibility of lead toxicity have 259 however, between neuronal and other cell types. been reviewed by Goyer and Mahaffey. Thus, a direct biochemical effect of lead on the Animals fed diets containing lower than recom-

-28 mended concentrations of nutrients generally retain difference in the development ofthe acoustic startle higher concentrations of lead in tissues than animals response was observed. Kimmel et al.,265 using a on normal diets.259 However, almost nothing is similar experimental design, also reported delays in known about the effects of elevating nutrient intake both surface and air righting in rats exposed to 50 or above recommended levels — which is the case with 250 ppm lead; and no differences were found in most commercial laboratory chows — on the tox­ either auditory startle, pinna detachment, eye open­ icity of lead. Additional research is needed in this ing, ear opening, or incisor eruption. area, and until further data are available on the in­ Sexual maturation appears to be one aspect of fluence of varying degrees of overnutrition, varia­ development that is quite sensitive to disruption by tion in nutrient intake must be suspected of altering lead exposure. Kimmel et al.265 reported a dose-re­ the toxicity of lead. lated delay in vaginal opening in female rats ex­ posed to 25, 50, or 250 ppm lead acetate in the 11.5.2.1 DEVELOPMENT drinking water starting at conception. In the group Several laboratories have reported on the effects exposed to 25 ppm lead, no differences in growth of perinatal lead exposure on physical, reproduc­ rates were observed. This suggests a direct effect of tive, and neurological development.247250260261 Un­ lead on sexual maturation rather than a change sec­ fortunately, in a number of studies either no control ondary to body weight changes. 250260262 was provided for undernutrition, or under­ Der et al.266 reported on the combined effect of nourished pair-fed controls showed similar develop­ parenteral administration of lead acetate and low 247 mental delays, thus masking any direct effects of protein diet (100 fig subcutaneously daily, ages 20 lead. to 61 days) on sexual development in the Sesco rat. Maker et al.263 also examined the effects of lead Lead significantly delayed the age at which vaginal exposure on brain development in mice. Two opening occurred in animals on the control diet. methods were used in an attempt to control for the Females given lead in combination with low protein effects of early undernutrition. In the first experi­ diets did not exhibit vaginal opening through 61 ment, two pair-fed litters were used. Pair-feeding days of age. The authors interpret these data on the was accomplished by allowing a litter access to an basis of a lead/protein-deficiency interaction. amount of normal chow equivalent to that consumed However, since animals were given 100 fig of lead by a litter on a 0.8 percent lead diet. Both pair-fed per day regardless of body weights and since their litters showed a reduction in brain weight at 30 days body weights were at 26 percent of control, the of age, as did the lead-treated animals. The two pair- dosage of lead per unit body weight was 400 percent fed litters, however, differed in the relative reduc­ greater than lead-treated animals on a control diet, tion in brain weight, one showing an identical which may account for the additional delay in response to the lead-treated groups and one showing maturation. brain weights intermediate between controls and Gray and Reiter261 studied the effects of lead ad­ lead-treated animals. A second attempt to alter the ministration (5 mg/ml in the drinking water at par­ nutritional state ofthe animals was accomplished by turition) on sexual maturation in the mouse. Vaginal altering litter sizes (3 pups versus 6 pups). Body opening was delayed about 4 days in lead-treated weights of control animals of the two litter size animals. No delay in development was seen in pair- groups were equivalent, however, so that varying the fed controls, further suggesting a primary effect of litter size over this range did not effectively produce lead on sexual maturation. Furthermore, no delay in undernutrition. Therefore, the conclusion of Maker sexual maturation was observed in animals when 263 et al. that underconsumption of food alone does lead was discontinued at weaning. Therefore, the not account for the slow development of litters on a presence of lead at the time of maturation appears lead diet is not supported by their data. essential for the lead-induced delay and may be re­ Reiter et al.264 examined development in rats ex­ lated to its effects on circulating hormones at the posed to lead both prenatally and during lactation time of puberty. via the mothers' milk. They reported a delay in both the age at eye opening and the age at development of 11.5.2.2 LOCOMOTOR ACTIVITY the air righting reflex in the 50 ppm treatment In the animal model, the most commonly group. This exposure level was shown to produce no employed behavioral index of lead toxicity has been depression in growth, which suggested a direct effect locomotor activity. As with other behavior, locomo­ of lead on nervous system development. No tor activity is influenced by a variety of factors that

11-29 P.74

include sex, age, time and duration of testing, type of important, but also of importance is the length of measurement, etc. The relative influence of these time over which the activity is measured. Short-term factors on the observed activity will vary with the measurements of activity in a novel environment experimental method employed. The endpoint being have been termed exploratory activity or locomotor measured is activity (not necessarily ambulation), reactivity and primarily reflect the animal's reaction and the nervous-system processes responsible for to the novel environment. This reactivity in turn will this activity and their relative contributions may be be affected by the structure of the environment. In different. Tapp,267 for example, compared seven order to determine spontaneous or basal activity different measures of activity in the rat and found levels, an animal must reside in an environment over virtually no intercorrelation. These results suggest long periods of time. Once the animal is established that the tests he employed were not measuring the in the environment, the activity levels of the animal, same behavior. Capobianco and Hamilton268 ex­ especially the rodent, will be highly dependent on amined the effects of various brain lesions on am­ the time of day. bulation as measured by three different methods: Therefore, in reviewing the lead literature, it must open-field, stabilimeter, and activity wheels. These be remembered that locomotor activity measure­ different measures of activity were affected ments do not represent a unitary behavior; careful differently by a given brain lesion. Lesions of the consideration must be given to the particular experi­ diagonal band, for example, produced increased ac­ mental methods employed. In addition, attempts to tivity in the running wheel, decreased activity in a extrapolate the results of an activity measurement in stabilimeter, and no change in activity in an open animals directly to the clinical situation are unwar­ field. ranted. A brief summary of pertinent studies is given Not only is the type of activity-measuring device in Table 11-5.

TABLE 11-5. EFFECTS OF LEAD EXPOSURE ON LOCOMOTOR ACTIVITY IN LABORATORY ANIMALS

Test conditions, in order o presentation: Lead concen­ 1. Method tration. ^1 % 2. Length of testing Nutritional Reference Species Exposure conditions Blood Brain 3. Group size status Results 4. Aje

Allen etal.*'* Monkey 0.5-9 mg/kg/day for 12 160-400 Observed locomotor Normal Hyperactivity weeks activity (qualitative measures) Bornschein etal.270 Mouse, (Charles 5 mg/ml lead acetate in 120-190 200-306 Proximity counter Undernourished Normal River) CD-I drinking water, starting 3 hours at parturition Individual 35 days

Brown"" Rat(Bar F • 35 mg/kg P.O. to dams Photoactometer Normal Rabbitry) from parturition to 21 20 min days Individual 49 days

Driscoll and Rat (Simonsen) 10* and 10" M lead Open-field Undernourished Hypoactive (61% of Stegner"" acetate in drinking water 2 min control) from conception Individual 31 days

1 Gray and Reiter"' Mouse, (Charles 5 mg/ml lead acetate in Residential maze Undernourished Hypoactive (75-80% of River) CD-I drinking water, starting 90-240 min control) at parturition Individual 30, 50,130 days

Hastings etal."'* Rat, (Long- 0.2 or 1.0 mg/ml from Control Running wheel Normal Normal Evans, parturition to 21 days = 11±4 3 weeks Charles 0.2 mg/ml Individual River) = 29±5 30-51 days 1.0 mg/ml = 42±4

Kostas etal."" Rat(Long-Evans) 0.05-5.0% lead acetate in Shuttle box, activity Normal - 0.05% group Hyperactivity in shuttle Blue Spruce mother's chow from wheel undernourished in box (182% of control). parturition until 21 days. 1 hr 5-5.0% Normal in activity Continued in offspring at Individual wheel. 0.25-25 ppm in chow 75-77 days; 90-93 until 35 days days 11-30 TABLE 11-5 (continued).

Test conditions, in order of presentation: 1. Method Lead concen­ 2. Length of testing tration. fig% 3. Group size Nutritional Reference Species Exposure conditions Blood Brain 4. Age status Results

Overman275 Rat, (Long- 10, 30, 90/mg/kg/day by 21 days Jiggle platform Normal Hyperactive Evans intubation from 3-21 90 mg/kg Four days Charles days of age = 226± 21 Individual River) 35 days 22-65 90 mg/kg = 56±5

Reiter"'" Rat, Sprague 5% lead carbonate in Jiggle cage; Undernourished Transient hyperactivity Dawley, mother's chow from Residential maze (200% of control at 13 Reiter etal."76 Charles River parturition to 16 days, 4 min; 14 days days). Normal at 44 50 ppm in drinking water Individual; group of 3 days. Normal levels in for remainder of 13-44 days; 120-160 adults; but disrupted experiment days ultradian rhythms.

Reiter et al.2'4 Rat, Sprague 5,50 ppm in drinking 180 day 180 day Residential maze Normal Hypoactivity (53-78% of Dawley, Blue water. 40 day males males 5 days controls) Cahill etal.27' Spruce pretreatment of parents. 0= 5± 0.5 0=18+ Groups of 3 Continued from 5= 6+ 0.4 50= 20± 2 120 days conception through 50= 10± 50= 27± 2 adulthood 0.6

Reiter etal.2"' Mouse, Charles 5 mg/ml lead acetate in Residential maze Undernourished Normal River, CD-I drinking water from 2 days parturition until 45 days Individual 100 days

Sauerhoff and Rat, Sprague 4% lead carbonate in 29 days Selective activity Undernourished Hyperactive at 29 days Michaelson278 Dawley, CO mothers' chow from 88± 1.1 meter (140190% of control) Sauerhoff27' parturition to 16 days 24 hours Normal at 50 days post-partum, 40 ppm in Groups of 6 drinking water 26-29, 50

Silbergeld and Mouse, 2, 5,10 mg/ml lead Proximity counter Undernourished Hyperactivity (300-400% Goldberg262.280.28i Charles River, acetate in drinking water 3 hours of control) CD-I starting at parturition Individual 30-150 days

Sobotka and Cook2"2 Rat, Sprague 8-91 mg/kg/day by 22 days Photoactometer Normal Normal and Sobotka et al.283 Dawley (Charles intubation from 3-21 81 mg/kg 30 min River) days of age = 71±12 Individual 35 days 24-28 days 81 mg/kg = 23±1.4

Sobotka etal.284 Dog (Beagle) 1 or 4 mg/kg/day orally Open field Not indicated Hypoactive from 2 weeks to 5 months Individual 3-4 months

Winneke etal.28' Rat (Wistar) 1.38 g lead acetate/kg of 16 days Open field Normal Hyperactive (129% of chow (745 ppm Pb) for 26.6 3 min, on consecutive control) 60 days pretreatment to 190 days • days mothers. Continued in 28.5 Individual offspring from 90-140 days -conception to testing

Zenicketal.2"" Rat, Sprague 750 or 1000 mg/kg/day to Open field Undernourished Initial hypoactivity in females on restricted 3 min, on 10 100 mg/kg group (days watering schedule from consecutive days 1 and 2) Hyperactive by 21-99 days of age. Individual the 7th test day. Exposure continued 22-32 days through gestation and weaning.

11-31 P.76

The data to be reviewed here suggest that Sauerhoff and Michaelson study,278 animals were perinatal lead exposure produces an altered reac­ rotated between testing cages, and, therefore, the ob­ tivity of an animal to a novel environment. Reac­ served increase in activity was consistent with a tivity is increased in the young animal, but this in­ lead-induced change in locomotor reactivity. In this creased reactivity disappears as the animal matures. experiment, pups were directly exposed by daily in­ In the adult animal, on the other hand, the lead ex­ tubation ranging from 10 to 90 mg/kg/day. This ex­ posure results in a reduced reactivity. As will be posure is similar to that used by Sobotka and seen, the exact nature ofthe change in locomotor ac­ Cook,282 who employed intubation levels of 9 to 81 tivity brought about by this altered responsiveness mg/kg/day, but who reported no differences in ac­ will depend heavily on the structure of the test en­ tivity in a photoactometer. vironment. One striking difference between these two experi­ Sauerhoff and Michaelson278 and Sauerhoff279 ex­ ments was in the reported blood lead levels, shown posed lactating females to lead using a modification in Table 11-6. Therefore, the higher internal ex­ of the Pentschew and Garro52 exposure regimen. posure seen in Overmann's experiment may have ac­ Litter mates were tested as a group at 25 to 28 days counted for the difference in the observed of age in a test case similar to the home cage. The behavioral effects, although differences in experi­ data were collected in four blocks of 3 hr and one mental protocol may also have accounted for block of 12 hr, extended over 4 days. Although they differences in the behavioral effects. are presented as counts per hour for a 24-hr period, data represent reactivity attributable to multiple TABLE 11-6. LEAD EXPOSURE AND RESULTING BLOOD LEAD LEVELS IN EXPERIMENTS MEASURING LOCOMOTOR short-term exposures to a novel environment, and, ACTIVITY IN RATS therefore, are not comparable with data obtained by continuous sampling over a 24-hr period. Offspring Blood lead.ug/dl Experiment 21 to 22 days 35 days ofa lead-exposed mother exhibited elevated activity 2 levels. Since only one group (n = 6 pups) was tested Sobotka and Cook 82 71 ± 12.4 23 ± 1.4 and Sobotka etal.2" from each treatment, however, no statistical test can (81 mg/kg/day) be applied to these data. Overmann275 258 226.1 ± 21.1 56 ± 4.6 Using a similar exposure regimen, Reiter and (90 mg/kg/day) Reiter et al.276 exposed animals to 5 percent lead carbonate in the chow starting at parturition. Two different laboratories have reported on the Animals were repeatedly tested at various ages, effects of lead administration on open-field activity beginning at 13 days of age, using a 4-min jiggle in 30- to 40-day-old rats. Driscoll and Stegner272 re­ platform activity measurement. This measuring ported a decreased activity in animals tested for 2 device detects both locomotor activity and station­ min. Zenick et al.286 tested animals for 3 min in an ary body movements. They reported an increased open field on 10 consecutive days. On the first 2 days activity (200 percent of control) in 13-day-old of testing, animals from the high exposure group animals. This elevated activity declined with age (1000 mg/kg/day in the drinking water of the and returned to control levels by 44 days of age. mother) showed a decreased activity similar to that Comparisons were made with pair-fed controls since reported by Driscoll and Stegner.272 By the seventh this exposure resulted in a significant growth impair­ day of testing, however, these animals were ambulat­ ment. Whether this return to normal levels was ing at a higher level than controls. These data can caused by maturation or by repeated testing was not also be interpreted as an increased locomotor reac­ determined in this study. Animals were also tested as tivity in lead-treated animals that is initially adults (120 days) in a residential maze, which allows manifested in an open field as decreased ambulation. continuous measurement of activity over extended With repeated exposure, the animals show an ele­ periods of time; this test showed no differences in the vated activity similar to that in previously reported activity levels as a result of treatment. However, the studies. The difference in the direction of lead-in­ ultradian rhythms of activity seen in control animals duced change in initial activity levels in these open during the nocturnal period (short-term, 4/hr field experiments as compared to the jiggle platform oscillation in activity) were absent in lead-exposed experiments258275 is probably a result of the animals. differences in the size of the test environments. The Overmann275 used a similar jiggle platform to larger open field results in decreased activity in measure activity in 22- to 65-day-old rats. As in the lead-exposed hyperreactive animals, whereas in the

11-32 smaller jiggle platform the animals show increased treated animals, but this difference disappeared as activity. the animals became established in the environment. Finally, Hastings et al.273 reported no differences On the other hand, activity levels remained sup­ in running-wheel activity of 30-day-old animals ex­ pressed during the nocturnal period. posed to lead by suckling with mothers receiving 1 In a second study, Reiter et al.276 reported no mg/ml lead in the drinking water. This exposure difference in residential-maze activity of adult lead- resulted in blood lead values of 42 figldl at weaning. treated animals. They speculated that the lack of a Since animals do not normally show much running lead effect on locomotor activity in the second ex­ activity upon initial exposure to the running wheel, periment may have been the result of the choice of ' the effects of this lead exposure on reactivity cannot animal supplier (Charles River). Since the Charles be adequately tested. These results do demonstrate River animals were normally less active in the maze, that long-term running wheel activity (3 weeks) was it may have been difficult to lower their activity not disrupted in these young, lead-exposed animals. further with treatment. Differences in experimental At or about the time of sexual maturation, lead protocol, i.e., differences in dose and period of ex­ exposure has not been shown to alter activity levels posure, however, also may have accounted for the in the rat. As previously indicated, Sauerhoff and differences in observed activity. Michaelson278 and Reiter et al.276 found no In summary, data on the rat suggest that perinatal differences in activity in animals tested between 44 exposure to lead may produce an increased reac­ and 50 days of age. These were animals which were tivity which disappears as the animal matures. This reported to have increased activity at a younger age. effect could result from a delay in normal matura­ Brown271 also reported no differences in locomotor tion of forebrain inhibitory systems.287 As the lead- activity in animals tested at 49 days of age either in treated animals mature, they pass through a period an photoactometer or in an open field. of normal reactivity which then progresses to a Available data also suggest that perinatal ex­ decreased reactivity in the adult animal. These data posure to lead may produce a decreased reactivity in would be consistent with a maturational lag seen in the adult animals. Kostas et al.274 measured locomo­ children288 and pose an interesting hypothesis that tor activity in adult rats exposed to either 0.05, 0.5, requires further testing. or 5.0 percent lead carbonate in the chow from par­ Sobotka et al.284 have reported decreased ambula­ turition until weaning and to 0.25, 2.5, or 25 ppm tion in young dogs exposed to lead and tested in a 7- from weaning until 35 days of age. Two measure­ by 7-ft open field. Allen et al.269 exposed infant ments of activity were employed. Animals tested for monkeys to lead via their formula and reported hy­ 1 hr in a shuttle box activity cage were found to have peractivity from 3 to 5 months of age. These data increased activity, whereas animals tested in running and the data of Sobotka et al.284 are consistent with wheels showed no difference from controls. This the lead-induced increase in reactivity seen in the lack of sensitivity of the running wheel to lead-in­ young rat. The data of Allen et al.269 must be duced changes in activity is consistent with the find­ qualified, however, since no quantitative measure of ing of Hastings et al.273 Again, the nature of the activity was made. change in shuttle box activity may be interpreted in In a series of often cited papers, Silbergeld and terms of a lead-induced decrease in reactivity Goldberg262-280-28' reported lead-induced hyperac­ toward a novel environment, since rats made more tivity in mice. Lactating females were exposed to reactive would tend to freeze in this environment, lead acetate in the drinking water, starting at par­ thus causing decreased ambulation. Winneke et turition, in concentrations of either 2, 5, or 10 al.285 found a similar change in reactivity as indi­ mg/ml. The authors reported a 300- to 400-percent cated by open-field activity scores over 5 successive increase in locomotor activity that extended from 30 days of testing. Lead-treated rats had significantly to 150 days of age in the offspring. The lack ofa con­ elevated activity on the first 3 days of testing, and trol for the growth retardation found in the iead- activity had returned to normal on days 4 and 5. treated groups makes interpretation of these data Reiter et al.264 reported a lead-induced decrease difficult. As previously indicated, Castellano and 256 in activity in adult animals tested in a residential Oliverio reported that early undernutrition pro­ maze. This test system allowed for measurement of duces hyperactivity in mice. Therefore, the observed various components of the animals' activity, includ­ hyperactivity may have resulted from the under­ ing exploratory, diurnal, and nocturnal activity. Ex­ nutrition, independent ofa lead effect. Greater con­ ploratory activity was initially suppressed in lead- cern, however, stems from the subsequent failure of

11-33 P.78

two different laboratories261-270289 to replicate the controls, which prevents the occurrence and the findings of Silbergeld and Goldberg, using the same recording of locomotor activity. strain of mouse and the same exposure regimen. Reiter258 examined the dose-response relationship Bornschein et al.270 exposed lactating mice to 5 to amphetamine in control, undernourished, and mg/ml lead acetate and tested offspring in activity lead-exposed rats. He found, as did Bornschein et chambers identical to those employed by Silbergeld al.,270 that undernutrition shifted the dose-response 262 280 281 and Goldberg. - - They were unable to verify curve to the left. Lead treatment, on the other hand, lead-induced hyperactivity in mice even though shifted the dose-response curve to the right. Thus, undernutrition was also present in their animals. under the appropriate conditions, lead exposure per Gray and Reiter261 were also unable to demon­ se can be shown to produce an attenuated response strate hyperactivity in lead-exposed mice, using a to amphetamine. The occurrence of a true paradoxi­ residential maze to measure activity. Furthermore, cal response, however, is questionable. 289 Reiter et al. were unable to find differences in the The lead-induced attenuated response to activity of mice experimentally exposed to lead from amphetamine has been observed regardless of birth to 45 days in Goldberg's laboratory and then whether the reported predrug activity levels were transported to the author's laboratory for behavioral elevated,280-281 normal,258-282 or depressed.264 The testing. determination of the exact nature of this altered Silbergeld and Goldberg280-281 also studied the response, i.e., altered CNS sensitivity versus altered locomotor response of lead-exposed mice to various absorption, distribution, and metabolism, requires drugs that are used in the treatment and diagnosis of further study. minimal brain dysfunction in children. Most nota­ bly, they reported that their lead-treated, hyperac­ tive mice responded paradoxically to the stimulants 11.5.2.3 LEARNING ABILITY amphetamine and methylphenidate. Examination of There is little doubt that acute, high-level lead ex­ the activity data following administration of posure in young children can produce overt 28 methylphenidate ' raises questions as to the exact manifestations of neurotoxicity.182-203 Mental retar­ nature of the paradoxical response. Both control dation is an established sequela of lead-induced en­ and lead-treated animals responded with increased cephalopathy in children.85-86 The extent and nature locomotor activity following 40 mg/kg of of lead-induced neurotoxicity following long-term methylphenidate; however, the response ofthe lead- low-level lead exposure during the developmental treated mice was markedly attenuated. Within 90 to years is also of continuing interest. As indicated pre­ 120 min, animals were below their predrug level. viously, restrospective studies in children are This time course in the response would be expected generally equivocal and are compromised by serious if the lead-treated animals were entering into experimental design limitations, e.g., unsatisfactory stereotypic behavior (a behavioral pattern char­ documentation of lead-exposure history prior to acteristically seen following high doses of behavioral testing and inappropriate or unsatisfacto­ 290 amphetamine-like compounds). Although the ry control groups.291 authors state that no stereotypic behavior occurred In an attempt to overcome some of these limita­ in lead-treated animals, no quantitation of this tions, investigators have turned to animal models of behavior was made. Furthermore, this stereotypic chronic, low-level exposure. The major portion of behavior has been observed in lead-treated mice by this work has been carried out in rats. Table 11-7 other investigators.270 Again, the possible contribu­ provides a summary ofthe pertinent studies, includ­ tion of early undernutrition to these results must be ing exposure conditions, testing conditions, and considered. Bornschein et al.270 found this paradoxi­ results. In an attempt to structure this literature and cal lowering of activity in undernourished mice facilitate evaluation, the organization shown in following 10 mg/kg of d-amphetamine, but only dur­ Table 11 -8 has also been developed. It separates the ing the second hour following drug administration. data along two lines: (1) tasks that reportedly are, or In the first hour, activity showed the expected in­ are not, sensitive to changes arising from the ex­ crease, although to a lesser extent than in controls. posure conditions and (2) the stage of learning dur­ These authors postulated that early undernutrition ing which effects are, or are not, demonstrable. The shifts the dose-response curve to the left such that acquisition column indicates investigations of the animals given high levels of amphetamine (10 rate at which animals form associations between mg/kg) enter into stereotyped behavior sooner than stimulus and reinforcement conditions. Measures

11-34 P.79

utilized to quantify the process are numbers of days of the quantitative nature of the behavior after the and trials or problem presentations required to desired criterion has been attained. The reversal/ex- reach some predetermined level of performance. tinction column contains studies of behavior ob­ The percentage of the test population attaining the served following the removal of (extinction) or defined criterion per unit time is also a common alteration of (reversal) the conditions that produce measure. The performance column includes studies reinforcement.

TABLE 11-7. PROTOCOLS USED FOR THE STUDY OF ANIMAL LEARNING

Behavioral tasl Lead exposure Number Litter Subjects Blood lead Mg/dl per test per test Grow1hd Learning Reference Species Apparatus Reward Task" Periodb Level" group group rate Peak Attest performance

Brady Rat water neg. Simult. PG.G.L 500 GAV 4 17 N 7 7 Impaired et al.»" T-maze BD. Brown, D.271 Rat T-maze pos. Succ. L 100 W ? 6 N 7 7 Impaired B.D. T-maze pos. Succ. L 25 GAV' 7 7 N 7 7 Impaired B.D. Tmaze pos. Succ. L(lst lOd) 35 GAV 7 5-6 N 46 7 Impaired B.D. T-maze pos. Succ. L(lastlld) 35 GAV" 7 5-6 N 20 7 No effect B.D. Tmaze pos. Succ. L(lastlld) 70 GAV 7 5-6 N 19 7 No effect B.D. T-maze pos. Succ. L(lastlld) 140 GAV 7 5-6 A 7 7 Impaired B.D. T-maze pos. Succ. Birth to 5 IP" 7 7 N 288 23 Impaired B.D. day 10 Brown, S. Rat water neg. Spatial Various 100 IP* 7 66 7 7 7 No effect et al."" Tmaze discrim. (8 days to 5 weeks) Shuttle- neg. 2-way 5 weeks 100 IP" 7 8 7 7 7 No effect box Driscoll and Rat Y-maze pos. Simult. PG.G.L.PW 2070 DT< 7 4 A 7 7 Impaired Stegner?"" B.D. Shuttle- neg. 2-way PG,G,L,PW 2070 DT' 7 5 A 7 7 Improved box Shuttle- neg. 2-way PG,G,L,PW 2070 DT' 7 12 N 7 7 Impaired box Shuttle- neg. 2-way PG,G,L,PW 2070 DT' 7 12 A 7 7 Improved box Overmann29* Rat E-maze pos. Spatial L 5,16,49 7 7 N 33,174 15,23, No effect discrim. GAV 226 56 E-maze pos. Tactile L 5,16,49 7 7 N 33,174 7 Impaired on discrim. GAV 226 reversal E-maze pos. Visual t 5,16,49 7 7 N 33,174 7 No effect discrim. GAV 226 No effect 2-compart neg. 1-way L 5,16,49 7 7 N 33,174 7 Impaired ment chamber GAV 226 2-compart- neg. Passive L * 5,16,49 7 7 N 33,174 7 No effect ment chamber avoid GAV 226 Operant pos. Temporal L 5,16,49 7 7 N 33,174 7 Impaired disc. GAV 226 Snowdon"95 Rat CF maze pos. Maze Adult 2.7,4.4 7 8-10 A 7 7 No effect learning exposure 6.6 IP" andPW CF maze pos. Maze L 4.4 IP« 17 36 A 7 7 Impaired learning Sobotka Rat Shuttle- neg. 2-way 3-21 days 5.15,44 6-7 28-34 7 11 7 Impaired etal.2«3 box post­ GAV 21 partum 23

(continued)

11-35 P.80

TABLE 11-7 (continued).

Number Lead exposure Blood lead, fig/d! Litter Subjects per test per test Growth Learning Reference Species Apparatus Reward Task" Penodb Level" group group rate Peak At lest performance

Sobotka and Rat 2- neg. passive 3-21 days 5,15,44" ? 5-8 18,61,71 No effect Cook"2 compartment avoid post­ GAV chamber partum Shuttle- neg. 1-way 7 5-8 18,61,71 No effect box Shuttle- neg. 2-way 5-8 18,61,71 Impaired box Operant neg. Spatial 5-8 18,61,71 Impaired discrim. reversal Slecta«6 Rat Operant pos. Fl PW (day 50,300 — 3 9,28,40 Altered schedule 22-57) 1000 W'

Shapiro Rat Operant pos. VI 30 PW (day .09 • 52 — Altered etal.2" schedule 90126) IP«

Hastings Rat Shock neg. Flinch 109, No effect etal.2" grid 546 W' 10 29,42 5,9 Shock neg. SEA 109, Less grid 546 W' 10 29,42 5,9 aggressive Operant pos. Succ. 109, 10 29,42 5,9 No effect B.D. 546 W'

Winneke Rat Lashley pos. Simult. PG.G.L 745 OT' — 10 29 29 Impaired et al.285 jumping PD&SD PW stand

Bornschein298 Mouse Operant pos. Simult. 103,546 5 15 A 79 29,120 Impaired B.D. 2730 W' -190 Bornschein2'" Mouse Operant pos. Simult. 109,546 7 14 N 80 29 Impaired B.D. W' 180 reversal Sobotka Dog Tmaze pos. Simult. 2-week- 1.4 W' — 10 N 85 — Impaired etal.284 R.O. 5 month post­ partum VanGelder Sheep Operant pos. Auditory Adult ex­ 100 GAV — Impaired etal.300 discrim. posure 9 weeks Operant pos. Fl 1000 DT' — 5 7 30 30 No effect schedule Operant pos. Fl 530 DT' — 5 7 17 17 No effect schedule Carson Sheep CF maze pos. Maze 2.3,4.5" — 6-8 7 17-25 914 No effect et al.30i.302 learning DT and VanGelder et al.soo CF maze pos. Maze 5 days- 2,4,8,16" — 4 7 57,81 — No effect learning 12 weeks DT 123,162 post partum Operant pos. Simult. G 2,3,4,5" — 6-8 7 17,25 9,14 Impaired FD&SD DT Bowman and Monkey WGTA pos. L.PL 0.3,1.0 — 4 — 50,85 50,85 Impaired Bushnell~43 reversal

"Succ. B.D. = Successive brightness discrimination. G= Gestation period, external exposure to dam. Simult. 8.D. = Simultaneous brightness discrimination. L= Lactation period, external exposure to dam. 1-way = One-way avoidance. PW- Post weaning period, external exposure to pup. 2-way = Two-way avoidance. CDT= Diet FD = Form discrimination. GAV° Gavage SD = Siie discrimination. IP= Intraperitoneal Flinch T= Tail-flinch test. W= Drinking water SEA = Shock elicited aggression. %= (formal CF mue = Close-field male. Operant = Operant chamber. A= Altered WGTA = Wisconsin General Test Apparatus. "mg/kg/day

bPG= Pregestation period, external exposure to dam. 'ppm

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A review ofthe studies listed in Table 1 1-7 leads little or no apparent rationale that would aid in the to the following general critique. Few animal studies formulation and testing of hypotheses and in­ adequately simulate the lead exposure conditions terpretation of data. Finally, most investigators do found in young children either with respect to the not provide adequate documentation of the relative levels of exposure or the timing of the. exposure. sensitivity of the behavioral tasks being used which There is often an inadequate or nonexistent history could be accomplished with the use of a standard of lead exposure with respect to both the external reference compound. Since this has not been done, and internal dose. In regard to the general experi­ failure to observe a disruption in task acquisition or mental designs, several design deficiencies fre­ performance could be taken to mean that (1) the task quently occur. These include: (1) limited sample size is not sensitive enough to detect the deficit or (2) the or larger samples derived from a few litters — the neural systems which mediate the behavior on that latter permits genetic effects to have an inordinate task are not affected by lead at the exposure levels influence on the results — and (2) inappropriate ap­ examined. plication of statistical methods and confounding of variables, e.g., maternal undernutrition or neonatal In spite of the extensive methodological growth retardation, which results in an inability to differences in these studies, several conclusions can determine specific causes for demonstrated effects. be drawn from the data shown in Tables 11-7 and The selection of behavioral tasks is often made with 11-8.

TABLE 11-8. STAGE OF LEARNING

Treatment effects Acquisition Performance Reversal 'extinction

Present Brady etal.292.a Shapiro etal.29™ Overmann294.a.b Brown304,b Slecta296.b Sobotka etal.283.a Carson etal.soi.b Bornschein et al.298-b Bornschein et al.298-1" Driscoll and Stegner272.a.b Bowman303.b Hastings etal.273b Overmann294.a Snowdon295-b Sobotka et al.283.a Winneke etal.285b Miller etal.sos.b

2 Absent Bornschein et al.298•*> Overmann 94.a Carson et al.soo.b Sobotka etal.283.a Hastings et al.273.b Miller etal.305.b Overmann29i a.b > Sobotka et al.283.a Winneke etal.285.b

aNegative reinforcer. bPosit ive reinforcer.

Although learning paradigms are being used to cers appear to be equally sensitive to disruption demonstrate effects of lead on CNS function, the following lead exposure (Table 11-8). Therefore, present data do not permit a clear distinction be­ lead exposure does not appear to have a selective tween the effects of lead exposure on cognitive func­ effect on a specific motivational system. tion (learning/memory) and effects on sensory- Treatment effects have been reported both by motor function, arousal, or motivation, which in those investigators using manual testing procedures turn can produce performance differences. which require a high degree of experimenter-subject Therefore, some of the studies appearing in the col­ interaction (cf. 285, 292, 304) and by those using umn titled "Acquisition" (Table 11-8) may, in fact, automated operant chambers with minimal experi­ belong in the "Performance" column. This is menter-subject interaction (cf. 283, 294, 306). Thus, especially true of studies that report large group reports of significant treatment effects cannot be differences on the first day of acquisition (cf. 292, ascribed to experimenter bias. 299). New studies specifically designed to test for this distinction must be conducted. The effects appear to persist beyond the immedi­ Tasks that use both positive and negative reinfor- ate exposure period with behavioral disruption

1-37 P.82

demonstrable in animals with normal blood lead behavioral testing paradigms will be necessary to levels (cf. 283, 285, 304, 306). resolve the conflict. Procedures have been inadequate for reproducing 11.5.2.4 EFFECTS OF LEAD ON AGGRESSIVE qualitative and quantitative changes in behavior. BEHAVIOR This has limited the ability to test hypotheses per­ taining to the site of action of lead-induced Two reports appeared in the literature in 1973 behavioral changes. which suggested that lead exposure produced an in­ 262 It is not yet clear whether the observed effects are creased aggressiveness. Silbergeld and Goldberg direct effects of lead on the developing nervous reported that mice exposed to either 5 or 10 mg/ml system or whether the effects are indirectly mediated of lead had a "heightened frequency of fighting as through treatment-induced alterations in maternal determined by the incidence of bite frequencies ob­ behavior, maternal milk, or one of several potential served on litter-mate males housed together." 278 peripheral target systems in the neonate. Sauerhoff and Michaelson also referred to an in­ Some types of learning problems appear to be creased aggressiveness in lead-exposed rats during more sensitive to lead-related disruption than the fourth week of development. In neither report, others. For example, in the passive avoidance however, was there an attempt to quantitate these paradigm, acquisition and extinction are reportedly observations of increased aggression. not sensitive.282-294 Simple pattern discrimination Hastings et al.306 exposed lactating rats to lead (0, is also apparently insensitive to lead expo­ 109, or 545 ppm) from parturition to 21 days. This sure. 285,294,301 Other forms of visual discrimina­ lead treatment produced no change in growth in the tion such as size285-301 and brightness272-292-304-306 ap­ offspring. Individual pairs of male offspring (from pear to be particularly sensitive. The two reports of the same treatment groups) were tested at 60 days of altered size discrimination285-301 may in fact be age for shock-elicited aggression. Lead-exposed special cases of brightness discrimination since the groups showed significantly less aggressive behavior different size stimuli (white pattern on a black back­ than the control group. There were no significant ground) also reflect different amounts of light. differences among the groups in the flinch/jump Further testing will be necessary to resolve this issue. thresholds to shock. This latter finding suggests that Active-avoidance tasks are also being used suc­ the differences seen in the shock-elicited aggression cessfully to examine effects obtained following lead were not caused by differences in shock threshold. exposure. Both one-way avoidance294-306 and two- Gray and Reiter261 reported on the aggressive way, shuttle-avoidance tasks272-283 reflect a disrup­ behavior of mice exposed to 5 mg/ml lead acetate tion in normal behavior. The one exception is a from parturition. Aggressive behavior was measured negative finding by Sobotka et al.283 using a one-way by introducing an adult male intruder into the home avoidance task. This negative effect may be related cage of an individual experimental male. Control to the fact that shock was terminated automatically males wounded the intruder 85 times on the average after 5 seconds, independent of the animals' during a 14-hour test period, whereas intruders to behavior. This is not the usual procedure, and its lead-treated and pair-fed male cages had means of effect cannot be evaluated since no data were pre­ 32 and 35, respectively. Therefore, the reduced ag­ sented for this particular task. gressive behavior seen in these experiments cannot Deficits in task performance do not appear to be be explained by lead exposure alone, because similar species specific since they are reported for reductions were observed in pair-fed controls. rats,272-283-294-304 mice,298 dogs,305 sheep,30' and Nevertheless, in both the rat and the mouse a quan­ monkeys.303 Furthermore, recent studies suggest that titative examination of aggressive behavior suggests behavioral alterations may be present in rats ex­ that lead can cause a decrease rather than an in­ posed to lead following weaning.296-297 These re­ crease in aggressive behavior. ports are contrary to the generally held opinion that adult or post-weaning rats are insensitive to lead ex­ 11.5.2.5 NEUROCHEMISTRY posure. 293.295.30i since the number of reported The effects of in vivo lead exposure on a variety of studies using adult exposure protocols is extremely neurochemical substances and processes have been limited, however, it is not possible to rule out the studied in the past several years (see Table 11-7). suggestion that these conflicting data merely reflect Perhaps most notable are the investigations of lead differences in task sensitivity. More research using effects on both putative neurotransmitter systems adult exposure protocols and more sophisticated and on energy metabolism in the central nervous

11-38 system. Much ofthe work, by its nature, has required transmitters and because of the documentation ex­ the use of experimental animal models for broad tant on the neurophysiological and behavioral roles screening for possible effects across many different played by these transmitters. The approaches transmitter systems or for testing specific hypotheses employed in these studies have included: (1) of altered transmitter functions. Unfortunately, biochemical assays of steady-state levels of transmit­ these studies on the neurochemical effects of lead ex­ ter substances in brain tissue;277-283-288-307"315 (2) posure are hindered by the same problems in experi­ assessment of synthesis and turnover rates;309-315 (3) mental design as those discussed in the section on measurement of the activity of enzymes responsible behavioral studies. for transmitter synthesis or degrada- Research on neurotransmitter systems has con­ tjon;283,309,3i 1,313,316-318 (4) assessment of transport centrated primarily on the effects of lead exposure processes involved in synaptic uptake of transmitters on cholinergic and monoaminergic functions, proba­ or their precursors;308-309-316 and (5) assessment of bly because of the extensive background literature synaptic release mechanisms308-309-315-316 (see Table that exists on the basic neurochemistry of those 11-9).

TABLE 11-9. IN VIVO EFFECTS OF LEAD EXPOSURE ON NEUROCHEMISTRY

Lead concentration Nutritional Blood. Brain. Species Exposure status Ittlil Mil Neurochemical parameter Silbergeld and Mouse 5 mg/ml lead acetate in Under­ 1) High affinity transport of 1) Decreased high affinity Goldberg308 drinking water starting at nourished phenylalanine, glycine, transport of dopamine parturition leucine, NE, 5HT, GABA, and choline DA, choline, and tyrosine Increased high affinity transport of tyrosine 2) Steady-state ACh NEDA 2) Increased steady state levels of NE Silbergeld etal.309 Mouse 5 mg/ml lead acetate in Under­ 1) ACh release 1) Decreased 40% drinking water starting at nourished 2) Steady state NE, HVA, 2) Increased 27, 41, 15% parturition VMA respectively 3) MAO 3) Increased 20% 4) Choline transport 4) Decreased 50% Carroll etal.3i* Mouse 2,5,10 mg/ml lead acetate Under­ 1)K+ -induced release of 1) Inhibited K+ induced in drinking water starting nourished ACh and choline release of choline and at parturition 2) Spontaneous release of ACh ACh 2) Increased spontaneous 3) Steady-state levels of ACh, ACh release choline 3) No change in steady state 4) CAT CBK AChE levels 4) No change Brown et al.318 Rat 7.5 mg/kg (IP) from birth Normal AChE No change in medulla to 10 days of age (regional brain analysis) oblongata, corpus striatum, cerebellum, cerebrum or hippocampus. Inhibited in midbrain (16%) Goiter and Rat 5% lead acetate in Under­ Steady-state NE, DA Increased NE at 33 days of Michaelson*"' mother's diet from par­ nourished age (13%); turition to 16 days post no change in DA partum, 40 ppm in drink­ ing water Intubated with 1.0 mg/day from parturition to 16 days post partum, 40 ppm in drinking water Michaelson and Rat 5% lead acetate in Under­ ? Control = 0.1 1) Steady-state DA 1) Decreased DA (20%) Sauerhoff*'* mother's diet from par­ nourished 4% = 0.88 2) Steady-state 5HT, GABA, 2) No change turition until day 16, 25 and NE ppm in drinking water Sauerhoff and Rat 4% lead carbonate in Under­ Steady-state NE, DA Decreased DA (20%) Michaelson"8 mother's diet from par­ nourished turition until 16 days, 40 , ppm in diet Grant et al."'" Rat 0, 25, 100 or 200 ? 0 = 16 0.15 Steady-state NE, DA No change mg/kg/day by gavage on 25 = 26 0.38 (regional brain analysis) postnatal days 3-25 100 = 43 0.51 200 = 63 0.68 (continued)

11-39 P.84 • r* •

TABLE 11-9 (continued).

Lead concentration Nutritional Blood. Brain, Reference Species Exposure status nW Hill Neurochemical parameter Results

Hrdina3l3 Rat 0.2 and 1.0 mg/kg IP 7 7 7 Cerebrocorticol ACh = 32-48% increase (lOOg)for 45 days ACh, AChE AChE - No significant change Brain stem NE NE - 20-27% decrease 5HT 5HT - No change Modak etal.3n Rat 1% lead acetate in drinking Under- Control = 0 7 1) Steady-state ACh 1)Increased ACh in dien­ water at parturition nourished 1%= 245 2) CAT cephalon (12%) 3)AChE 2) Increased in medulla- pons, hippicampus and cerebral cortex (11-12%) 3) Lower in medulla-pans, midbrain and dien­ cephalon (10-20%) Sobotka etal."8" Rat 8-91 mg/kg/day by intuba­ Normal 8 71 — 1) Steady-state NE, DA, 5HT 1) No change tion from 3-21 days of age 2)AChE 2) Decreased (marginal) ShihandHanin3i5 Rat 4% lead carbonate in Under­ ? 7 1) Steady-state ACh, choline 1) No change in ACh, in­ mother's chow from par­ nourished 2) ACh turnover creased choline turition to 21 days, 40 2) Decreased ACh turnover ppm in diet (33-51%) Cahill etal."" Rat 5, 50 ppm in drinking Normal 180 day 180 day Steady-state NE, DA Decreased DA at 28 days of water. 40 day pretreat­ 0= 5 0= 18 age ment of parents continued 5=6 5= 20 Increased NE at 180 days of from conception through 50 = 10 50= 27 age adulthood Silbergeld and Mouse 5 mg/ml lead acetate in Under­ 7 7 Brain HVA and VMA Increased 33 and 48% Chisolm3i9 drinking water starting at nourished parturition Gerber et al.312 Mouse 0.1-1000 mg/l lead acetate ? ? 7 Steady-state 5HT No change in drinking water for one year

Bhatnagar3i7 Rat 0,1,2% lead acetate for 70 •> 7 7 Tyrosinase activity No effect (200- days 250 g) Bull etal."" Rat 1) 67 /xM in vitro lead — — — K+ stimulated respiration Inhibition (200- chloride of cerebral slices 400 2)3,12, 60 mg/Pb/Kg total Impaired Control = 0.08 0.06 Inhibited at 12 and 60 g) dose over 2 weeks growth in 60 3= 13.2 0.17 K+ stimulated respiration mg/kg exposure level mg/kg group 12= 72.6 0.41 of cerebral slices 60= 380 1.02 Holtzman and Hsu"""1 Rat 4% lead carbonate at 2 Under­ 7 7 Cerebral and cerebellar Impaired respiration after 2 weeks postpartum nourished, mitochondrial respiration weeks of treatment reduced brain weights

Abbreviations: ACh • Acetylcholine CAT • Choline acetyltranferase AChE • Acetylcholinesterase NE - Norepinephrine DA - Dopamine 5NT • 5-riydroiytrypta mine (serotonin) GABA • Gamma aminobutyric acid CPK - Choline phosphokinase

11-40 Several studies utilizing high levels of lead ex­ by Wender et al.,321 urinary metabolites reflect pri­ posure have reported inhibition of cholinergic func­ marily peripheral nervous system activity. In tion. In a series of experiments on the mouse, another study322 altered levels of 5-hydroxyindole Silbergeld et al.308-309 and Carroll et al.316 reported acetic acid (5-HIAA) were reported in the urine of decreased potassium-induced release of occupationally lead-exposed battery factory acetylcholine (ACh) and decreased high-affinity workers, suggesting possible lead effects on transport of choline. Unfortunately, no control for serotonin (5-hydroxytryptamine) systems. No growth retardation was provided in these experi­ parallel supportive evidence from animal studies has ments and relatively high external exposures to lead been advanced for such an effect, however, and in were required to produce the effect. This was also fact most reports claim negative findings for any true in the rat studies reported by Modak et al.311 type of measurements of brain serotonin func­ and Shih and Hanin.315 At lower levels of external tion. 283,308,312-314 exposure, with no accompanying growth retarda­ The reasons for the inconsistencies of lead-in­ tion, no consistent effects on cholinergic function duced changes in monaminergic and cholinergic have been reported (cf. 283 and 318). Thus, if im­ functions may be the result in part of interlaboratory paired cholinergic function during in vivo lead ex­ differences in dosing regimens and other variations posure is also found in the absence of undernutri­ in experimental protocol. One note of caution, tion, and/or growth retardation, which is probable in however, is appropriate here in that highly variable view ofthe in vivo work reported later in this section, results are seen within different laboratories, even then its relevance to behavioral effects seen at lower with the same exposure regimens, assay procedures, exposure levels will need examination. etc., from experiment to experiment. This might sug­ The effects of lead exposure on catecholamine gest that some subpopulations of rats or mice might function have also been extensively studied. Find­ be resistant to lead effects on monoamine transmit­ ings have been reported of increased steady-state ters whereas others are more vulnerable, possibly levels of norepinephrine,277-307-308 increased activity because of genetic factors, subtle variations in diet, of monamine oxidase (MAO),309 increased synap­ etc. More carefully controlled studies in the future tosomal transport of the precursor tyrosine,308 and that explicity manipulate such variables (genetics, increased amounts of the norepinephrine metabolite, nutrition, etc.) may reveal lead effects even at low vanillyl mandelic acid, and homovanillic acid in the exposure levels, given the right circumstances or brain.319 In studies on steady-state levels of population segment tested. norepinephrine, changes have been reported either Finally, the recent report of Nathanson and in the absence of undernutrition227 or when values Bloom323 indicated that in vitro lead exposure in­ 307 have been compared to pair-fed controls. How­ hibits adenyl cyclase activity (I50 = 2.4 fiM). This ever, inconsistencies within a given laboratory (cf. enzyme is responsible for the synthesis of cyclic 278 vs. 307), absence of similar findings in different adenosine 3',5'-monophosphate (c-AMP) which has laboratories (cf. 283 and 310), and findings of de­ been shown to play an important role in the mechan­ creased steady-state levels313 make any conclusions ism of action of a number of hormones, including regarding lead-induced changes in noradrenergic neurotransmitters. The effects of lead exposure on systems equivocal. This uncertainty is also found in adenyl cyclase and the resultant effects on neuro­ the reports on dopamine changes following lead ex­ transmitter systems warrant further investigation. posure (cf. 277, 278, 314 vs. 283, 307, 310). Several recent reports have dealt with the effects Human studies attempting to relate subclinical pf lead exposure on brain-energy metabolism. Bull lead exposures to signs of altered brain monoamine et al.253 reported that both in vivo and in vitro lead function have been initiated utilizing urinary levels exposure inhibited potassium-stimulated respira­ of monoamine neurotransmitter metabolites as in­ tion. Of interest was the finding that lower brain dices of CNS monoamine turnover rates. Although levels of lead (approximately 1 /30th) were required initial studies on catecholamine excretion have sug­ to inhibit respiration in vivo than in vitro. Also, these gested a lead effect, ah appended note by Silbergeld results were seen in animals showing normal growth and Chisholm319 indicated difficulty in finding one (12 mg/kg group). Similar findings of inhibited of the earlier reported effects in subsequent studies. respiration using isolated mitochondria were re­ This clinical study again emphasizes some of the ported by Holtzman and Hsu320 and by Brierley.324 problems and uncertainties that have beset in­ In contrast to the general lack of consistent effects vestigations of low-level toxicity. Also, as indicated on steady-state levels of cholinergic substances and

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associated enzymes, consistent evidence for effects but unfortunately, no data were presented by of lead on cholinergic synaptic uptake and release Silbergeld et al.329-330 on the muscle contraction mechanisms have been reported. following electrical stimulation, so the relative im­ More specifically, lead resembles other divalent portance of this finding cannot be evaluated. cations in that it appears to interfere with chemically Finally, Cooper and Steinberg331 demonstrated that mediated synaptic transmission as demonstrated by lead is also capable of blocking neural transmission studies of peripheral neural functions. Kostial and at the adrenergic synapse. They measured the con­ Vouk325 reported that in vitro perfusion of the cat traction force of the rabbit saphenous artery follow­ superior cervical ganglion with 4.8 fiM lead nitrate ing stimulation of the sympathetic nerve endings. depressed or blocked nerve transmission. Contrac­ Again, the results indicated that lead blocks muscle tion of the nictitating membrane during contraction by an effect on the nerve terminals acetylcholine perfusion was unaltered. Also, perfu­ rather than an effect on the muscle. Since the sion of the ganglion with excess calcium restored response recovered when calcium concentration was acetylcholine release and thus reversed the lead increased in the bathing solution, it was concluded blockade. From these findings Kostial and Vouk that lead does not deplete transmitter stores in concluded that lead depressed synaptic transmission the nerve terminals but more likely blocks by impairing acetylcholine release from the pre­ norepinephrine release. synaptic terminals. In summary, in vitro experiments have demon­ Manalis and Cooper326 and Cooper and strated that lead interferes with synaptic transmis­ Manalis327 showed that lead can influence both pre- sion in the peripheral nervous system. This effect ap­ and postsynaptic events. Using the frog (Rana ip- pears to be related to a competitive inhibition piens) sciatic-nerve/sartorius-muscle preparation in of calcium-mediated, evoked release of the vitro, they demonstrated that the principal effect of neurotransmitters. Further, lead was shown to in­ lead was on presynaptic transmitter release, crease the spontaneous release of transmitter from although lead had a weak, curare-like effect on the some synapses. postsynaptic response to applied acetylcholine. They The effects of lead on synapses within the CNS 325 confirmed the findings of Kostial and Vouk that have not been extensively studied. Carroll et al.,316 lead depresses the phasic release of transmitter however, reported a decrease in potassium-induced evoked by nerve stimulation. They further observed release of both choline and acetylcholine from corti­ that lead increases spontaneous release of cal minces of mice chronically exposed to lead. acetylcholine as evidenced by increased miniature These changes in acetylcholine metabolism suggest end-plate potentials (MEPP's). These MEPP's that, as is the case of the peripheral nervous system, represent the response ofthe postsynaptic membrane the central cholinergic function may be depressed by to released acetylcholine in quantities that are in­ lead. This is further supported by a report of Shih sufficient to depolarize the membrane to threshold and Hanin315 that lead exposure decreased in vivo levels. In a subsequent experiment Kober and acetylcholine turnover rate in cortex, hippocampus, 328 Cooper demonstrated that in the frog, lead blocks midbrain, and striatum (35, 54, 51, and 33 percent synaptic transmission in the sympathetic ganglion by decreases, respectively) in rat brain after neonatal competitive antagonism of spike-evoked entry of lead exposures. Along with the in vitro findings, this calcium into the presynaptic nerve terminals with a provides additional evidence supportive of lead-in­ resultant reduction in acetylcholine release. duced dysfunctions of cholinergic synaptic uptake Experiments by Silbergeld et al.329-330 indicated a and release mechanisms. Unfortunately, these similar blockade of transmitter release by lead in the results and many from the above peripheral function rat. Furthermore, they reported a reduced force of studies were obtained at rather high exposure levels contraction in the. phrenic-nerve/diaphragm pre­ and most were performed on undernourished or paration from mice exposed to lead from birth growth-retarded animals. through 60 days of age. The nerve-muscle prepara­ One final note concerns the relationship between tion from these lead-exposed animals showed a levels of lead in blood and brain. Several studies on reduction in force of contraction with nerve stimula­ rodents have reported simultaneous lead values for tion. Also, a reduced force of contraction was re­ blood and brain resulting from lead exposures of ported upon direct stimulation of the muscle. This various durations. Bornschein et al.270 exposed mice observation agrees with the weak postsynaptic to 5 mg/ml lead acetate starting at parturition. effects of lead reported by Manalis and Cooper;326 Brain/blood ratios showed a steady increase from 20

11-42 to 100 days of age. Ratios of 1.05, 2.55, and 4.08 perinatal exposure to lead produces an increase in were reported for mice at 20, 40, and 100 days of the animal's behavioral reactivity. Ifthe test condi­ age, respectively. This increase in the brain/blood tions are appropriate, this increased reactivity will ratios resulted from both a steady increase in brain be manifested as increased locomotor activity, lead levels (increasing from 200 fig % at 20 days of although some text situations will show reduced ac­ age to 584 /ug % at 100 days of age) and a decrease tivity. Therefore, the altered activity levels per se in blood lead levels (decreasing from 190 ppm at 20 are merely reflective ofa more basic nervous system days to 143 ppm at 100 days). dysfunction. Close scrutiny of the available data Cahill et al.277 reported blood and brain levels of would also suggest that altered locomotor activity in lead in rats exposed from conception to either 0, 5, young animals occurs only at moderately high ex­ or 50 ppm lead in the drinking water. At parturition, posure levels. A comparison of the data of Sobotka brain/blood ratios of offspring were 0.91 and 0.5 for and Cook282 and Overmann275 are of interest in this exposure levels of 5 and 50 ppm, respectively. At regard (see Table 11-4). Using gastric intubation, 180 days of age, these ratios were 3.3 and 2.7, Overmann reported hyperactivity in young rats respectively. A ratio of approximately 1 was also re­ whose 21-day blood lead levels were 226 figldl. ported by Grant et al.310 in 30-day-old rats exposed Sobotka and Cook, using a similar exposure regi­ to various levels of lead from 3 to 25 days of age. men, found normal activity levels with 22-day blood These data are consistent with the results of lead levels of 71 figldl. It appears, therefore, that Bornschein et al.270 and indicate that initially this early change in reactivity occurs at fairly high brain/blood ratios are approximately unity but that blood lead levels. It may be, then, that the changes with continued exposure the ratios steadily increase. in activity currently reported in laboratory animals are more representative of a post-encephalopathic 11.5.2.6 SUMMARY AND CONCLUSIONS hyperactivity than of subclinical effects as has been Data obtained in laboratory animals, such as that suggested. 52 reported in the rat by Pentschew and Garro, indi­ On the other hand, the reactivity changes reported cate that encephalopathy is produced by high-level in older animals with lifetime exposure occur at perinatal exposure to lead. This encephalopathy oc­ much lower blood levels (cf. 264, 277, and 285). curs to varying degrees in different species and is characterized by the relative involvement of Finally, reports on the effects of lead exposure on neuronal degeneration and vasculopathy. the acquisition and/or performance of operant It seems clear that with regard to CNS toxicity the responses indicate that perinatal exposure to moder­ developing organism represents the population at ate and low levels of lead may disrupt this behavior. greatest risk. Whether this increased risk is at­ Thus, external exposures that result in blood lead tributable to a greater sensitivity or to a greater levels ranging from 30 to 80 figldl have been re­ susceptibility of the developing organism will re­ ported to disrupt cognitive function (see Table quire further testing. That is, with a given external 11-5). As is true with the clinical data, this area re­ exposure, the CNS of the developing organism quires further investigation. reaches a higher concentration of lead and is It has been repeatedly indicated that serious therefore more susceptible to poisoning. Whether methodological problems exist in the animal the threshold for a given effect of lead is lower in the literature which make it difficult to interpret many immature nervous system versus the adult will need of'the available data. Future research in this area to be determined. would benefit from more tightly controlled experi­ There is also good evidence that perinatal ex­ ments including: posure to lead even at moderate exposure levels will 1. Better documentation of internal exposure, produce delays in both neurological and sexual not only with respect to lead levels but also development. Because these effects have been dem­ with respect to correlative indices of lead tox­ onstrated to occur in the absence of either under­ icity, e.g., ALAD, protoporphyrin, etc. nutrition or growth retardation, it has been sug­ 2. The use of exposure protocols that do not pro­ gested that they represent direct effects of lead in the duce confounding variables such as under­ respective organ systems. nutrition, differences in both litter size and In the animal studies, locomotor activity has been number of litters per treatment group, etc. the most commonly used behavioral index of lead 3. Validation of behavioral tests using both toxicity. The data reviewed here suggest that positive control substances and cross corre-

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lation with other physiological indices. This most marked in children with encephalopathy. The would include research aimed at more closely aminoaciduria disappears after treatment with defining both the relative sensitivity of the chelating agents and clinical remission of other CNS compared to other organ systems as well symptoms of lead toxicity.85 This is an important ob­ as the contribution of other indirect actions of servation relative to the long-term or chronic effects lead on the resulting behavioral changes. of lead on the kidney. 11.6 EFFECT OF LEAD ON THE RENAL In a group of children with slight lead-related SYSTEM neurological signs, generalized aminoaciduria was found in 8 of 43 children with blood lead levels of 11.6.1 Acute Effects 40 to 120 /ig/dl.99 It should be noted that the More than 60 years ago an English toxicologist, children reported to have aminoaciduria in the study 99 Thomas Oliver,332 distinguished acute effects of lead of Pueschel were not specifically identified as to on the kidney from lead-induced chronic nephropa­ their lead exposure. Thus, it is not possible to state thy. Acute renal effects of lead are seen in persons what level of lead exposure within the blood lead dying of acute lead poisoning or suffering from lead- range of 40 to 120 ug/dl was associated with the induced anemia and/or encephalopathy and are effects. A similar renal tubular syndrome has been 336 usually restricted to nonspecific degenerative reported to occur in industrially exposed adults. changes in renal tubular lining cells, usually cloudy swelling, and some degree of cellular necrosis. Cells 11.6.2 Chronic Effects of the proximal convoluted tubules are most There is convincing evidence in the literature that 333 severely affected. As long ago as 1928, Pejie prolonged lead exposure in humans337 can result in emphasized that the degenerative changes in prox­ chronic lead nephropathy. Cramer et al.337 in 1974 imal tubules, rather than the vascular changes often reported on a group of 7 lead-exposed workers who referred to in earlier studies, are primary evidence had been exposed up to 20 years. Aminoaciduria was of injury to the kidney in lead poisoning. Many sub­ not found, and inulin clearance and renal blood flow sequent studies have shown at least three pathologi­ were also reported normal. The average blood lead cal alterations in the renal tubule with onset during level was 100 ug/dl, the minimum was 71 ug/dl, and the early or the acute phase of lead intoxication in all had strikingly high urinary ALA excretion. Some the kidney. These include the formation of inclusion with very long exposures were reported to have in­ bodies in nuclei of proximal tubular lining cells and terstitial and peritubular fibrosis, determined by the development of functional as well as ultrastruc­ renal biopsy. This pathological finding is commonly tural changes in renal tubular mitochondria. referred to as chronic lead nephropathy, which is Dysfunction of proximal renal tubules (Fanconi's characterized by slow development of contracted syndrome) is manifested by aminoaciduria, kidneys with pronounced arteriosclerotic changes, glycosuria, and hyperphosphaturia, and was first fibrosis, glomerular atrophy, and hyaline degenera­ noted in acute lead poisoning by Wilson and tion of these vessels. This is a progressive disease, coworkers in 1953.334 Plasma amino acids were nor­ sometimes resulting in renal failure. It seems to oc­ mal, which suggested that the aminoaciduria and cur sporadically, primarily in industrially exposed other functional abnormalities were of renal origin. workers and in older adults who have been diag­ Subsequently, aminoaciduria in children with acute nosed as having lead poisoning early in life. There is lead poisoning was observed by Marsden and also some evidence that it occurs in long-time Wilson335 in England, and Chisholm85-86 found that drinkers of lead-contaminated whiskey, as reported 338 9 of 23 children with lead encephalopathy had by Morris et al. in the cases of 16 adults treated aminoaciduria, glycosuria, and hypophosphatemia. over a 10-year period. This study reported lead Aminoaciduria was seen more consistently in poisoning manifested by the same symptoms found Chisolm's studies than the other two manifestations in children. The most specific pathological change of tubular damage. Thus, the amino acid transport reported was the presence of large numbers of acid- system is probably more sensitive to the toxic actions fast intranuclear inclusions within the cells of the 339 of lead than the transport systems for glucose and kidney tubules and liver. Ball and Jorenson re­ phosphate. The aminoaciduria was generalized in ported a high frequency of saturnine gout resulting that the amino acids excreted in greatest amounts from the consumption of lead-contaminated were those normally present in urine. The condition whiskey, convincingly demonstrating reduced renal was related to severity of clinical toxicity and was uric acid clearance associated with plumbism.

11-44 In a series of 102 cases of lead poisoning studied and 17 of these had hypertension and/or by Lilis et al.',340 18 cases of clinically verified albuminuria. In a more recent study, Emmerson346 chronic nephropathy were found. For the whole presented a criterion for implicating lead as an series, the mean blood lead level was approximately etiological factor in such patients: the patients 80 ug/dl, with a range of 42 to 141 ug/dl. should have an excessive urinary excretion of lead Nephropathy was more common among patients following administration of calcium EDTA. In his who had been exposed to lead for more than 10 study, 32 patients with chronic renal disease at­ years than among those who had been exposed for tributable to lead poisoning had similarly elevated less than 10 years. In both studies, reduced urea excretion of lead. The presence of intranuclear in­ clearance preceded reduced creatinine clearance. clusion bodies is very helpful in establishing a rela­ In the Danilovic study,341 7 of 23 cases had blood tionship between renal lesions and lead toxicity, but lead levels of about 100 to 200 ug/dl. In the studies inclusion bodies are not always present in persons of Albahary et al.,342 blood lead levels were not re­ with chronic lead nephropathy. ported but exposure levels must have been quite high Attempts to confirm the relationship between because the mean ALA excretion was about 37 childhood lead intoxication and chronic nephropa­ mg/24 hr for 29 workers. These studies indicate that thy have not been successful in at least two studies in the nature of the effect is glomerulovascular, with the United States. Tepper90 found no evidence of reduction in clearance of urea and, in more chronic renal disease in 42 persons with a well-docu­ protracted exposures, also of endogenous creatinine. mented history of childhood plumbism 20 to 35 Also, reduced clearance of uric acid was observed in years earlier at the Boston Children's Hospital. 342 the study of Albahary et al. Likewise, Chisolm347 found no evidence of renal dis­ In the recently reported studies of Wedeen et ease in 62 adolescents known to have had lead intox­ al.,343 eight subjects suspected of excessive occupa­ ication 11 to 16 years earlier. An important distinc­ tional exposure were given detailed examinations tion between the Australian group and patients in for renal function. Four of the subjects showed signs the United States was that none of Chisholm's347 of abnormal renal function. In one subject with subjects showed evidence of increased residual body asymptomatic renal failure, chelation therapy in­ lead burden following the EDTA mobilization test. creased the glomerular filtration rate, the p-amino This difference has suggested to Chisholm that lead hippurate (PAH) extraction, and the maximal PAH toxicity in the Australian children must have been of excretion rate, and improved the proximal tubule a different type, with a more protracted course than ultrastructure, despite decreased renal plasma flow. that experienced by the American children. Most Three ofthe subjects showed proximal tubule abnor­ children in the United States who suffer from lead malities via biopsy. In eight subjects, lead-induced toxicity do so early in childhood, between the ages of nephropathy was established by exclusion. The 1 and 4, the source usually being oral ingestion of blood lead values ofthe individuals ranged from 48 flecks of wall paint and plaster containing lead. ug/dl, for the subject having asymptomatic renal failure, to 98 ug/dl. The lead levels ofthe other two 11.7 REPRODUCTION AND DEVELOPMENT subjects in the preclinical renal dysfunction category As reviewed thus far in the present chapter, the ad­ were 51 and 66 ug/dl. All subjects showed glomeru­ verse effects of lead on the hematopoietic, nervous, lar filtration rates of less than 87 ml/min/1.73m2. and renal systems have been well documented across The authors suggest on the basis ofthese studies that a wide range of exposure levels and represent a triad lead nephropathy may be an important occupational of symptoms classically associated with lead poison­ hazard in the U.S. lead industry. ing. Extensive evidence for adverse effects of lead on A series of reports from Queensland, Australia,344 reproduction and development has also been ac­ points to a strong association between severe lead cumulating in the literature for many years and has poisoning in childhood including central nervous become a matter of increasing medical concern. system symptoms and chronic nephritis in early Data from both human and animal studies indicate adulthood. Henderson345 followed up 401 children that lead exerts gametotoxic, embryotoxic, and, who had been diagnosed as having lead poisoning in possibly, teratogenic effects that impact on the pre- Brisbane between 1915 and 1935. Ofthese 401 sub­ and postnatal survival and development ofthe fetus jects, 165 had died, 108 from nephritis or hyperten­ and newborn, respectively. In addition, it appears sion. This is greatly in excess of expectation. Infor­ that the viability and development ofthe fetus may mation was obtained from 101 ofthe 187 survivors, also be markedly affected by lead indirectly via ad-

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verse effects on various health parameters, e.g., In a more recent study351 ofthe pregnancies of 104 nutritional state or blood chemistry, ofthe expectant Japanese women married to lead workers before and mother. The vulnerability of the fetus to such lead after their husbands began lead work, miscarriages effects while in utero has contributed to concern that increased to 84.2/1000 pregnancies from a prelead pregnant women may be a special group at risk for rate of 45.6/1000. The miscarriage rate for 75 lead poisoning. Certain information on adverse lead women not exposed to lead was 59.1/1000. effects on male reproductive functions, it should Another recent study by Fahim et al.352 in humans also be noted, has led to additional concern regard­ suggests that subtoxic lead absorption during preg­ ing the impact of lead on men. nancy may be associated with an increased incidence of preterm delivery and early membrane rupture: 11.7.1 Human Studies 253 women delivered in Rolla, Missouri (Region Data suggesting that lead exerts adverse effects on I), which is 60 to 80 miles from lead smelters, and human reproductive functions have existed in the 249 women delivered in Columbia, Missouri literature since before the turn of the century. For (Region II), where there is no lead industry. The in­ example, Legge,348 in summarizing the reports of 11 cidence of term pregnancies with early membrane English factory inspectors in 1897, found that of 212 rupture was 17 percent in Region I and 0.41 percent pregnancies in 77 female lead workers only 61 living in Region II. The incidence of premature deliveries children were produced. Fifteen workers had never was 13.04 and 3 percent, respectively. A high cor­ become pregnant. There were 21 stillborns, miscar­ relation was found between lead concentrations in riages occurred 90 times, and, of 101 children born, maternal and fetal blood: both were significantly 40 died in the first year. Legge also noted that when higher in the cases of preterm pregnancies and early pregnant animals were fed lead they always aborted. membrane ruptures than in term pregnancies. He concluded that maternal exposure to lead Pregnancy is a stress that may place a woman at resulted in a direct action ofthe element on the fetus. higher risk for lead exposure. Both iron deficiency 180 In 1911, Oliver published statistics in Britain and calcium deficiency increase the susceptibility of on the effect of lead on pregnancy (Table 11-10) lead toxicity, and women have an increased risk of which showed that the miscarriage rate was elevated both deficiencies during pregnancy and postpartum. among women employed in industries in which they The cause of the increased perinatal mortality may were exposed to lead. be a mutagenic or teratogenic effect of lead.353 TABLE 11-10. STATISTICS ON THE EFFECT The above studies clearly demonstrate an adverse OF LEAD ON PREGNANCY"" effect of lead on human reproductive functions, Number of Number of ranging from reduced pregnancy rates to increased abonions and neonatal deaths incidence of miscarriages, premature deliveries, stillbirths per (first year) per Sample 1000 females 1000 females and stillbirths. The mechanisms underlying these effects are unknown at this time. Many factors could Housewives 43.2 150 Female workers (mill work) 47.6 214 contribute to the above results, ranging from lead Females exposed to lead effects on maternal nutrition or hormonal state premaritally 86.0 157 before or during pregnancy to more direct Females exposed to lead gametotoxic, embryotoxic, or teratogenic effects after marriage 133.5 271 that could affect fertility or fetal viability during Since the time of the above studies, women have gestation. Efforts have been made to define more been largely excluded from occupational exposure precisely the points at which lead may affect to lead. Even before the effects of industrial lead ex­ reproductive functions both in the human female posure on pregnancy were documented, however, and male, and in other animals, as reviewed below. lead compounds were known for their embryotoxic In regard to potential lead effects on ovarian func­ properties and were often used to induce criminal tion in human females, Panova354 reported a study abortion.349 In a study by Lane,350 women exposed of 140 women working in a printing plant for less to lead levels of 75 ug/m3 were examined for effects than 1 year (1 to 12 months) where ambient air on reproduction. Longitudinal data on 15 pregnan­ levels were <7 ug lead/m3. Using a classification of cies indicated an increase in the number of stillbirths various age groups (20 to 25, 26 to 35, and 36 to 40) and abortions. No data were given on urinary lead in and type of ovarian cycle (normal, anovular, and women, but men in this sample had urinary levels of disturbed lutein phase), Panova claimed that 75 to 100 ug/liter. statistically significant differences existed between

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the lead-exposed and control groups in the age range preservation were controlled for contamination. of 20 to 25 years. It should be noted that the report Wibberly and coworkers359 have recently found does not show the age distribution, the level of sig­ that placental lead levels in the case of stillbirth or nificance, or the data on specificity of his method of neonatal death were significantly higher than in the classification. Also, Zielhuis and Wibowo,355 in a case of normal births. Placental levels were greater critical review of the above study, concluded that than 1.5 ug/g in only 7 percent ofthe normal births, study design and presentation of data are such that it whereas levels were greater than this in 61 percent of is difficult to evaluate the author's conclusion that the stillbirths or neonatal deaths. This does not chronic exposure to low lead in air leads to a dis­ mean that lead is a causal factor in such deaths and turbed function of ovaries. It should be noted that could indicate that lead accumulates in the placenta no consideration was given to the dust levels of lead, in times of fetal stress. an important factor in print shops. There are a number of recent studies on the Unfortunately, little else besides the above report passage of lead through the placental barrier as exists in the literature in regard to assessing lead assessed by lead levels in cord blood and/or mater­ effects on ovarian function or other factors affecting nal blood. For example, in the study of Gershanik et human female fertility. Nor are there many studies al 360,361 98 cord-blood samples matched with offering firm data on maternal variables, e.g., hor­ maternal blood samples showed a high correlation monal state, that are known to affect the ability of between lead levels in infants (mean = 10.1 ug/dl) the pregnant woman to carry the fetus full term. In and their mothers (mean = 10.3 figldl), with a addition, there are no studies that demonstrate con­ product moment correlation coefficient of 0.6377. clusively a direct lead-induced teratogenic effect on This suggests that infants may be born with blood the human fetus, although the transfer of lead across lead levels that essentially match those of their the human placenta and its potential threat to the mothers. In regard to assessing groups at risk for conceptus have been recognized for more than a cen­ such prenatal exposure, these authors also studied a tury.356 Nevertheless, documentation of placental group of 218 cord-blood samples (170 urban, 48 transfer of lead to the fetus and data on relevant rural) and observed that the mean urban value (9.7 parameters, e.g., fetal blood lead levels resulting ug/dl) was significantly different (p <0.05) from the from such transfer, help to build the case for a po­ mean rural value (8.3 ug/dl), suggesting a higher tential, but as yet not clearly defined, threat for sub­ risk of urban newborns for prenatal lead exposure. tle teratogenic and other deleterious health effects. Similarly, Scanlon362 sampled cord-blood randomly The placental transfer of lead has been estab­ from normal infants whose mothers had suburban (n lished, in part, by various studies that have disclosed = 15) or urban (n = 13) residences. The average ur­ measurable quantities of lead in human fetuses or ban value was 22.1 figldl (10 to 37 ug/dl), whereas newborns. An analysis of human fetal tissues by the corresponding suburban level was 18.3 ug/dl. Barltrop357 demonstrated that placental transfer of Smoking was without significant effect on these lead began as early as the 12th week of gestation and levels. These results tend to confirm the findings of 361 363 that total lead content increased throughout fetal Gershanik et al. Harris and Holley, on the development, with the highest concentrations occur­ other hand, surveyed cord and maternal blood in 24 ring in bone, kidney, and liver, and lesser, but sig­ pairs (11 suburban and 13 urban) and found a mean nificant, amounts occurring in blood, brain, and cord-blood value of 12.3 ug/dl and a mean maternal heart. Barltrop has also pointed out that the dis­ blood level of 13.2 ug/dl. No significant difference tribution of lead within the fetus at different stages was therefore seen in cord-blood levels as a function of development is probably more important than the of maternal residence, though a larger sample might total amount present at birth. have yielded significant effects since the ones found Of interest in this regard are the data of Schroeder were in the same direction as those found in other and Tipton,358 who showed that the mean lead level studies. in brains of stillborn U.S. children (n = 22) was 10 That the prenatal exposure of the fetus to lead, ppm (dry ash), but that there was an undetectable even in the absence of teratogenic effects, may be of level for normal infants, young children, and teens consequence in regard to adverse health effects is (0 to 19 years; n = 23). In this study, however, the demonstrated by studies relating fetal and cord­ levels of detection are not stated, so that the relative blood levels to some changes in fetal heme synthesis increase in level cannot be assessed. Also, it is not and claimed incidences of premature births. Haas et clear to what extent fetal tissue preparation and al.364 examined 294 mother-infant pairs for blood

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lead levels as well as for the corresponding urinary direct in that tests for hypothalamopituitary in­ ALA levels. The maternal blood mean was 16.89 fluence were negative. A group of 150 workmen who figldl and the fetal blood mean was 14.98, with a had long-term exposure to lead in varying degrees correlation of 0.538 (p <0.001). In the infants, the was studied. Clinical and toxicological criteria were levels of blood lead and urinary ALA were used to categorize the men into four groups: lead- positively correlated (r = 0.1877, p <0.0I). poisoned workmen (74.5 ug/dl) and those showing Whether a biological significance exists here, moderate (52.8 figldl), slight (41 figldl), and however, is not clear. According to the authors,364 physiologic (23 ug/dl) absorption of lead. Semen the positive correlation between lead in blood and analysis revealed asthenospermia, hypospermia, and urinary ALA for the group as a whole indicated teratospermia in lead-poisoned workers and those there was already an effect at lower blood lead with moderately increased absorption oflead (blood levels, i.e., increased susceptibility of heme syn­ lead levels = 50 to 80 ug/dl for the latter). The ab­ thesis. normal spermatozoa included binucleated, In a study of Fahim365 on cord-blood lead levels, bicephalus, amorphous, and tapered forms. In con­ blood lead values in pregnant women having pre­ trast, slightly increased or physiologic absorption of term delivery and premature membrane rupture, lead had no effects on the reproductive ability of and residing in a lead belt area (mining and smelting workmen. area), had significantly higher blood lead levels than In the review of Stofen,369 data from the work of women delivering at full term. A confusing aspect of Neskov in the USSR were reported involving 66 this study, however, is the similarity of blood lead workers exposed chiefly to lead-containing gasoline levels in women in the nonlead and lead belt areas. (organic lead). In 58 men there was a decrease or Though a number of other problems may be seen disappearance of erection, in 41 there was early with the analytical aspects of this study, it must be ejaculation, and in 44 there was a diminished num­ noted that among the 249 pregnant women in the ber of spermatocytes. control group outside the lead belt area the percen­ The literature reviewed here on lead effects on tage of women having preterm deliveries and pre­ human reproduction and development leaves little mature rupture were 3 and 0.4 percent, respectively, doubt as to the fact that lead does, in fact, exert sig­ whereas the corresponding values for the lead area nificant adverse health effects on reproductive func­ (n = 253) were 13.04 and 16.99 percent, respec­ tions. Most studies, however, have typically looked tively. at the effects of prolonged moderate-to-high ex­ With reference to more subtle prenatal effects, posures to lead, e.g., those encountered in industrial Palmisano et al.366 noted failure to thrive and situations, and many reports do not provide definite neurological deficits in a 10-week-old infant whose information on external exposure levels or blood mother had lead poisoning concomitant with lead levels at which specific effects are observed. alcoholism during pregnancy. When this infant was 11.7.2 Animal Studies challenged with a chelating agent, an abnormal urin­ ary excretion of lead was observed, indicating in­ Animal experiments have demonstrated that trauterine exposure. Postnatal exposure in this case levels of lead that are compatible with life have in­ was ruled out. Other, more controlled laboratory terfered with normal reproduction. Many studies studies on animals (discussed later) also suggest that assessed the effects of lead exposure of both parents teratogenic effects occur, but usually only at very on reproduction. Schroeder and Mitchener,370 for high lead exposure levels. example, showed a reduction in the number of A report367 on fatal birth defects in children con­ offspring of rats and mice that were given drinking ceived during a period of time when their father was water containing lead in a concentration of 25 ppm. 371 lead poisoned hints at important effects of lead on In a subsequent report, however, it was noted that the fetus being mediated via human males as well as animals in the earlier study were chromium defi­ females. Certain other studies369-370 demonstrated cient. No effects were found in animals with normal likely lead effects on various aspects of male diets. The combined effect of maternal and paternal reproductive functions. oral lead intoxication upon reproductive perfor­ 372 Lancranjan et al.368 have reported that mance was studied in rats by Morris et al. who re­ moderately increased lead absorption (blood lead ported significant reduction in weaning percentage mean = 52.8 ug/dl) resulted in gonadal impair­ among offspring of rats fed 512 ppm lead. Stowe and 373 ment. The effects on the testes were shown to be Goyer assessed the relative paternal and maternal

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effects of lead as measured by the progeny of F, of their dams and were mated in combinations as lead-toxic rats. Sprague-Dawley female rats being follows: control female to control male (CF-CM), fed laboratory chow with and without 1 percent lead control female to lead-toxic male (CF-PbM), lead- acetate were bred to normal, mature, Sprague- toxic female to control male (PbF-CM), and lead- Dawley males. The pregnant rats were continued on toxic female to lead-toxic male (PbF-PbM). The their respective rations with and without lead results identifying specific deleterious paternal and throughout gestation and lactation. Offspring of maternal effects of lead toxicity upon rat reproduc­ these matings, the F: generation, were fed the rations tion are shown in Table 11-11.

TABLE 11-11. REPRODUCTIVE PERFORMANCE OF F, LEAD-TOXIC RATS***-**

Type of mating Parameter CF-CM CF-PbM PbF-CM PbF-PbM

Litters observed 22 24 36 16

Pups/litter 11.90 ± 0.40a 10.10 ± 0.50 8.78 ± 0.30b 7.75 ± 0.50= Pup birth weight, g 6.74 ± 0.15 5.92 ± 0.13c 5.44 ± 0.13=."" 4.80 ± 0.19C-MJ Weaned rats/litter 9.84 ± 0.50 7.04 ± 0.77<= 5.41 ± 0.74C1 2.72 ± 0.70cd.e Survival rate, % 89.80 ± 3.20 73.70 ± 7.90 52.60 ± 7.20 30.00 ± 8.20C.-U Litter birth weight/Dam breeding weight, % 28.04 ± 1.30 22.30 ± 0.90= 19.35 ± 1.00c 15.38 ± 1.10C-U Litter birth weight/Dam whelping weight, % 19.09 ± 0.80 15.97 ± 0.58= 14.28 ± 0.66c 11.58 ± 0.78=--".'

Gestation gain/Pups per litter, g 11.54 ± 0.60 11.20± 0.74 11.17 ± 0.54 12.34 ± 1.24

Nonfetal gestational/Gain/fetus, g 3.93 ± 0.38 4.83 ± 0.47 4.15 ± 0.42 3.96 ± 0.46

aMean ± S.E.M. bSignificantly (p <0.05) less than mean for CF-CM. ^Significantly (p <0.01) less than mean for CF-CM. -"Significantly (p <0.01) less than mean for CF-PbM. eSignificantly (p <0.01) less than mean for PbF-CM. 'Significantly (p <0.05) less than mean for PbF-CM.

The paternal effects of lead included a 15 percent lead acetate at doses of 5 and 100 ug for 30 days. reduction in the number of pups born per litter, a 12 Control females possessed the same levels of lead percent reduction in the mean pup birth weight, and concentration in their blood as those for male con­ an 18 percent reduction in pup survival rate. The trols. However, for the treated animals the blood maternal effects oflead included a 26 percent reduc­ lead levels for the females were higher than those for tion in litter size, 19 percent reduction in mean pup the males: 30 figldl versus 19 ug/dl at 5 ug lead ace­ birth weights, and 41 percent reduction in pup sur­ tate, and 53 ug/dl versus 30 ug/dl at 100 ug lead vival. The combined male and female effects of lead acetate. They noted impotence and prostatic hy­ toxicity resulted in 35 percent reduction in the num­ perplasia in the males at the lower dose, progressing ber of pups per litter, 29 percent reduction in the to testicular damage with inhibition of sper­ pup birth weights, and 67 percent reduction in pup matogenesis in those reaching blood levels of 50 survival to weaning. Stowe and Goyer373 classified ug/dl. In the females, they noted irregularity ofthe the effect of lead upon reproduction as gametotoxic, estrus cycle at both doses. When lead levels reached intrauterine, and extrauterine. The gametotoxic 50 ug/dl, the female rats developed ovarian follicu­ effects of lead appear to be irreversible and had ad­ lar cysts with a reduction in the number of corpora ditive male and female components. The in­ lutea. A subsequent study employing Sprague- trauterine effects resulted from the transmammary Dawley rats was unable to replicate these find­ passage of lead from the dam to the suckling pup ings.375 adding insult to the gametotoxic and uterine en­ A number of other studies have focused more vironmental effects. specifically on either maternal or paternal lead ex­ The effects of lead on the reproduction of sexually posure effects. For example, in reference to maternal mature male and female Sesco rats were reported by effects, histopathological changes in ovaries in lead- Hildebrand et al.374 The animals were orally fed poisoned Rhesus monkeys have been demon-

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strated.376 Most other animal studies have utilized cidence of pregnancy, indicative of fertility, was rodents. 52.7 percent in the control group as compared to Kennedy et al.377 administered an aqueous solu­ 27.6 percent in the treated group. The fertility ofthe tion oflead to mice (days 5 to 15 of gestation) and to treated males was reduced to 50 percent. They rats from days 6 to 16 of gestation. At dosage levels calculated the mutagenicity index (number of early fetal deaths/total implants) to be 10.4 for lead- of 7.14, 71.4, and 714 mg/kg body weight there were 2 no observed effects on the number of fetuses treated mice versus 2.9 for controls (X = 10.4, p < 0.05). resorbed or the number of viable fetuses. No 379 teratogenic effects on gross examination were seen, In the study of Maisin et al., male mice received and an effect on body weight was observed only at 0.1 and 1 percent lead, as the acetate, in the diet. the highest level employed (714 mg/kg). The percentage of abnormal spermatozoa increased with increasing exposure. Ultrastructural changes Hubermont et al.378 exposed female rats to lead in were present. drinking water (0.1, 1, and 10 ppm) for 3 weeks 369 In the review of Stofen, several studies from before mating, during pregnancy, and 3 weeks after Russian laboratories were evaluated. As cited by delivery. In the highest exposure group (10 ppm), St6fen, Egorova et al., for example, injected lead at maternal and newborn blood and kidney lead values a dose of 2 ug/kg 6 times over a 10-day period and were elevated. Inhibition of 8-ALAD and elevation observed damage to testes and spermatozoa. Stofen of FEP in tissues were also noted. also reported that Golubova et al. found morpho­ 379 Maisin et al. exposed female mice to lead in the logical changes in testes of rats that received 2 mg diet (0.1 and 0.5 percent) from the day of vaginal lead/kg but not in rats receiving 0.2 mg/kg. plug to 18 days afterwards. The number of pregnan­ Lead appears to be teratogenic in some species, at cies decreased and the number of embryos succumb­ least at high exposure levels. McClain and ing after implantation increased. Becker,385 for example, administered single doses of Similarly, Jacquet380 exposed female mice via 25 to 70 mg/kg of lead nitrate intravenously to preg­ lead in diet (0.125, 0.25, and 0.50 percent) from nant rats on days 8 through 17 of gestation. A vaginal plug to 16 to 18 days afterwards. At the mid­ urorectocaudal syndrom of malformations was pro­ dle dosage, pregnancy incidence decreased, the duced when lead was administered on the 9th day of number of embryos dying before implantation in­ gestation. The lead nitrate was increasingly embryo- creased, and the number of corpora lutea showed a and fetotoxic when administered on later days of decrease. At the highest dosage, the number of gestation (days 10 to 15) but not teratogenic. Ferm 386 387 embryos dying after implantation increased, and Carpenter as well as Ferm and Ferm re­ whereas decreases in body weight of surviving ported increased embryonic resorption and malfor­ embryos were seen. mation rates when various lead salts were ad­ ministered to pregnant hamsters on the 8th day of Other studies have focused on lead effects on gestation. The teratogenic effect of lead was almost paternal reproductive functions. For example, the completely restricted to the tail region. Malforma­ 381 382 data from studies of rabbits, guinea pigs, and tions ofthe sacral and caudal vertebrae, resulting in 373 383 rats - indicate that paternally transmitted effects absent or stunted tails, were observed. from lead can occur, including reductions in litter size, in weights of offspring, and in survival rate. The reasons for the localization ofthe teratogenic effects of lead are unknown at this time. Ferm and Cole and Bachhuber,381 using rabbits, were the Ferm387 have suggested that the specificity could be first to confirm experimentally the paternal effects explained by an interference with specific enzymatic of lead intoxication. The litters of dams sired by events during early development. Lead alters lead-toxic male rabbits were smaller than those mitochondrial function and enhances or inhibits a sired by control males. Weller382 similarly demon­ variety of enzymes,388 any or all of which could in­ strated reduced birth weights and survival among terfere with normal development. Ferm389 has also offspring of lead-toxic male guinea pigs. reported that in the presence of cadmium the Verma et al.384 fed a 2-percent aqueous solution teratogenic effect of lead in hamsters is potentiated. of lead subacetate in drinking water to 14 male Swiss Studies by Giliani390-391 show that lead is mice for 4 weeks. The total mean intake of lead teratogenic to chick embryos. When 2-day-old amounted to 1.65 g. They placed the male with 3 embryos were given varying doses of lead acetate virgin untreated females for 1 week. The overall in­ (0.005 to 0.08 mg/egg) and were examined on the

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8th day of incubation, congenital cardiac anomalies firm statements on exposure levels at which any such were demonstrated.390 The incidence of cardiac effects on the fetus from maternal or paternal lead anomalies rose with increasing doses of lead. Other burdens might be observed. important anomalies were reduced body size, micromelia, shortened neck, microophthalmia, rup­ 11.8 THE ENDOCRINE SYSTEM tured brain, shortened beak, twisted neck and limbs, The endocrine effects of lead are not well defined and everted viscera.391 The most common develop­ at the present time. Lead is known, however, to mental anomalies were retarded growth and neck decrease the thyroid function in man and experimen­ abnormalities. It should be noted that in these tal animals. Porritt393 suggested in 1931 that lead studies high, acute doses of lead were administered. dissolved from lead pipes by soft water was the cause There is a paucity of information regarding the of hypothyroidism in individuals living in southwest 394 teratogenicity and developmental toxicity of chronic England. Later, Kremer and Frank reported the lead exposure. Kimmel et al.265 exposed female rats simultaneous occurrence of myxedema and plumb­ 395 chronically to lead acetate via drinking water (0.5, ism in a house painter. Monaenkova in 1957 ob­ 131 5, 50, and 250 ug/g) from weaning through mating, served impaired concentration of I by thyroids in gestation, and lactation. No teratogenic effects were 10 out of 41 patients with industrial plumbism. Sub­ 396 13I observed, although exposure to 250 ug/g lead ace­ sequently, Zel'tser showed that in vivo I uptake tate caused a slight but nonsignificant increase in and thyroxine synthesis by rat thyroid were fetal resorptions. The lead-treated animals pro­ decreased by lead when doses of 2 and 5 percent duced litters of normal numbers, but the offspring lead acetate solution were administered. Uptake of 131 from the 50- and 250-ug/g groups weighed less at I, sometimes decreased in men with lead poison­ weaning and showed delays in physical develop­ ing, can be offset by treatment with thyroid-stimulat­ 397 398 ment. Reiter et al.264 have also observed delays in ing hormone (TSH). - Lead may act to depress the development of the nervous system in offspring thyroid function by inhibiting SH groups or by dis­ 397 exposed to 50 ug/g lead throughout gestation and placing iodine in a protein sulfonyl iodine carrier, lactation. Whether these delays in development and the results suggest that excessive lead may act at result from a direct effect of lead on the nervous both the pituitary and the thyroid gland itself to im­ system of the pups or reflect secondary changes pair thyroid function. (resulting from malnutrition, hormonal inbalance, Sandstead et al.399 studied the effects of lead in­ etc.) is not clear. Whatever the mechanisms in­ toxication on the pituitary and adrenal function in volved, these studies suggest that low-level, chronic man. There was a decrease in secretion of pituitary exposure to lead may induce postnatal developmen­ gonadotrophic hormones. Their data suggested that tal delays in rats. lead may interfere with pituitary function in man It should be noted that the above reports on nor­ and may produce clinically significant hypopituitar­ mal developmental delays might be analogous to ism in some. Its effects on adrenal function were less certain suggested neurobehavioral effects of lead consistent, but some of the patients showed a from in utero exposures of humans (Section 11.5). In decreased responsiveness to an inhibitor addition, it has been demonstrated392 that con­ (metapyrone) of 11-beta-hydroxylation in the syn­ centrations of lead of approximately 170 ug/dl thesis of Cortisol. whole blood can inhibit 8-ALAD activity in both Excessive oral ingestion of lead in man has blood and brain of suckling rats. Although brain resulted in pathological changes in the pituitary- tissue was not purged of residual blood. ALAD con­ adrenal axis as indicated by decreased metapyrone tribution to brain ALAD activity would not be ex­ responsiveness, a depressed pituitary reserve, and pected to be significant. It is possible that 8-ALAD decreased immunoreactive ACTH.400-401 These activity might be diminished in utero at these lead same events may also affect adrenal gland function levels and that lead at these levels might have inasmuch as decreased urinary excretion of 17-hy- harmful consequences on neurological development droxycorticosteroids was observed in these patients. ofthe fetus. There is need for more critical research Suppression of responsiveness to exogenous to evaluate the possible subtle toxic effects of lead to ACTH in the zona fasciculata of the adrenal cortex the fetus. This overall evaluation in the offspring has been reported in lead-poisoned subjects,402 and may need to be correlated with the possible additive impairment of the zona glomerulosa ofthe adrenal effects of paternal lead burden. At this time, cortex has also been suggested.403 however, insufficient evidence exists to allow for There also is some evidence suggesting that lead

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may cause a derangement in serotonin metabolism oflead exposure on drug metabolism in children and or utilization. Tryptophan is the precursor of the adults. There were no differences between two nor­ neuroendocrine regulatory amine, serotonin. An mal children and eight lead-poisoned children in effect of lead on serotonin synthesis or utilization is their capacities to metabolize two test drugs, anti­ inferred in part from the observation of Urbanowicz pyrine and phenylbutazone. This might suggest that et al.404 who reported a rise in 5-hydroxyindole low plasma concentrations of lead do not have an acetic acid (5-HIAA) excretion in the urine of effect on the hepatic cytochrome P-450-dependent workers heavily exposed to lead. This rise preceded enzymatic activities in children. In 2 acutely the rise in urinary 8-ALA and coproporphyrin. A poisoned children, in whom plasma levels of lead similar rise in 5-HIAA excretion was noted in exceeded 60 figldl, antipyrine half-lives were sig­ moderately lead-exposed workers.322 More re­ nificantly longer than normal, and therapy with cently, however, Schiele et al.,405 using a different EDTA led to biochemical remission of the disease analytical method, were unable to find any signifi­ and restoration of deranged drug metabolism cant elevation in 5-HIAA excretion. toward normal. Hepatic drug metabolism in eight adult patients 11.9 THE HEPATIC SYSTEM showing marked effects of chronic lead intoxication The effect of lead poisoning on liver function has on the erythropoietic system was studied by Alvarez not been extensively studied. In a laboratory study et al.4" The plasma elimination rate of antipyrine, of 301 workers in a lead smelting and refining which, as noted above, is a drug primarily facility, Cooper et al.406 found serum glutamic ox­ metabolized by hepatic microsomal enzymes, was alacetic transaminase (SGOT) activity at an in­ determined in eight subjects prior to and following creased value of 11.5 percent in subjects with blood chelation therapy. In seven of eight subjects, chela­ lead levels below 70 ug/dl, 20 percent in those with tion therapy shortened the antipyrine half-lives, but a blood lead level of about 70 ug/dl, and 50 percent the effect was minimal. The two authors concluded in workers with a blood lead level of about 100 that chronic lead exposure results in significant in­ ug/dl. The correlation between blood lead levels hibition of the heme biosynthetic pathway without and SGOT was not statistically significant. In the ab­ causing significant changes in hepatic cytochrome sence of information on the possible influence of P-450-associated enzymatic activities. diet, infection, or personal habits, however, the authors were unable to draw any definite conclu­ 11.10 THE CARDIOVASCULAR SYSTEM sions concerning the etiology of these changes. Under conditions of long-term exposure at The liver is the major organ for the detoxification high levels, arteriosclerotic changes have been of drugs. In acute lead poisoning, the mixed-func­ demonstrated in the kidney. In 1963, Dingwall-For­ tion oxidase system of liver endoplasmic reticulum dyce and Lane82 reported a marked increase in the is impaired.407 The activity of this enzyme system, cerebrovascular mortality rate among heavily ex­ involved in the hepatic biotransformation of posed lead workers as compared with the expected medicaments, hormones, and many environmental rate. These workers were exposed to lead during the chemicals, is closely related to the availability ofthe first quarter of this century when working conditions microsomal hemoprotein, cytochrome P-450.408 It were quite bad. There was no similar increase in the has been shown that in rats lead induces inhibition of mortality rate for men employed more recently. heme synthesis and, therefore, causes a reduction in Hypertension is an important element in the cytochrome P-450 levels, with consequent impair­ etiology of cerebrovascular deaths. Tabershaw and ment ofthe mixed-function oxidase system.409 Drug- Cooper412 did an epidemiological study of 1267 metabolizing activities were significantly decreased workers who had been exposed to lead as a result of in the lead-poisoned animals. Intensity and duration their occupation in either the battery or lead smelt­ of these changes were dose dependent. In vivo ex­ ing industry between 1947 and 1970. Many were periments, based on the duration of pentobarbital found to have blood lead concentrations in excess of sleeping time, provided further evidence for the in­ 80 ug/dl. The authors concluded that there was ex­ hibition of drug metabolism in lead-poisoned rats. cess mortality associated with only two categories of These data would suggest than an enhanced sen­ illness, chronic nephritis and hypertension. The in­ sitivity to xenobiotics (drugs, pesticides, food addi­ creased incidence of hypertension in lead workers tives, etc.) should be expected to occur in lead- has also been reported by Monaenkova and poisoned animals. Alvarez et al.410 studied the effect Glotova413 and Vigdortchik.414 On the other hand,

11-52 Cramer and Dahlberg415 studied the incidence of hy­ change in the permeability of blood vessels (first, in­ pertension in a population of 364 industrially ex­ creased permeability; second, decreased per­ posed men, 273 of whom had a long-term exposure meability) in rats, rabbits; and dogs that received a to lead. They subdivided the workers into lead- solution of lead acetate. A phase change in the con­ affected and nonlead-affected groups based on the tent of catecholamines in the myocardium and in the urinary coproporphyrin test. There was no blood vessels was observed in subacute lead poison­ statistically significant difference between the ing in dogs.425 This effect appears to be a link in the groups nor was the incidence higher than that ex­ complex mechanism ofthe cardiovascular pathology pected for nonexposed men in the general popula­ of lead poisoning. tion. Other reports on the question do not show hy­ 11.11 THE IMMUNOLOGIC SYSTEM pertension to be unduly prevalent among lead workers.350-416 It is not clear, therefore, whether the Recent reports suggest that exposure to lead may vascular effects of lead in man are direct effects on interfere with normal susceptibility to infection. blood vessels or whether the effects are secondary to Hemphill et al.426 found that mice injected with, renal effects. subclinical doses of lead nitrate for 30 days showed There are conflicting reports regarding whether greater susceptibility to challenge with Salmonella lead can cause atherosclerosis in experimental typhimurium than controls that received a saline in­ 427 animals. Sroczynski et al.417 observed increased jection containing no lead. Selye et al. found that serum lipoprotein and cholesterol, and cholesterol rats injected with lead acetate (minimal effective deposits in the aortas of rats and rabbits receiving dose of 1 mg/100 g body weight) were susceptible to large doses of lead. On the other hand, Prerovska,418 a variety of bacterial endotoxins (toxins produced using similar doses of lead given over an even longer by the bacteria themselves) to which this species is period of time, did not produce atherosclerotic le­ ordinarily resistant. Administration of lead acetate sions in rabbits. in drinking water to male mice from 4 weeks of age Structural and functional changes of the myocar­ to sacrifice at 9 to 12 weeks old increased the toxic dium have been noted in children with acute lead response of the mice to 5 classes of viruses against 428 429 poisoning, but, to date, the extent of such studies has which it was tested. - These viruses were an been very limited. Cases have been described in RNA picornavirus (encephalomyocarditis), a DNA adults and in children, always with clinical signs of herpesvirus (pseudorabies). an RNA leukemia virus poisoning. There is, of course, the possibility that the (Rauscher leukemia), and RNA arbovirus B (St. coexistence of lead poisoning and myocarditis is Louis encephalitis), and an RNA arbovirus A coincidental. In many cases in which encephalopa­ (western encephalitis). thy is present, the electrocardiographic abnor­ Among the factors that may be involved in pro­ malities disappeared with chelation therapy, sug­ ducing this decreased resistance to infection is the gesting that lead may have been the original etiologi­ decreased production of antibodies. Williams et 430 cal factor.419421 Silver and Rodriguez-Torres421 al. reported that lead binds antibodies in vitro and noted abnormal electrocardiograms in 21 of 30 could potentially do so in vivo. Chronic exposure to children (70 percent) having symptoms of lead tox­ mice of lead acetate in drinking water produced a icity. After chelation therapy, the electrocar­ significant decrease in antibody synthesis, particu­ 431 diograms remained abnormal in only four (13 per­ larly gamma globulins. cent) of the patients. Electron microscopy of the Phagocytosis (ingestion of foreign material by a myocardium of lead-intoxicated rats has shown cell specialized for that purpose) by alveolar diffuse degenerative changes.422 In a review of five macrophages is believed to be an important step in fatal cases of lead poisoning in young children, the removal of dust particles and bacteria from the degenerative changes in heart muscle were reported respiratory tract. Consequently, the activities of to be the proximate cause of death.91 It is not clear alveolar macrophages are important aspects of that such morphological changes are a specific pulmonary defense. Bingham et al.432 found that the response to lead intoxication. Kosmider and continuous inhalation of lead sesquioxide aerosol 3 3 Petelnz423 examined 38 adults over 46 years of age (10 ug/m to 150 ug/m ) by rats for 3 to 12 months with chronic lead poisoning. They found that 66 per­ significantly reduced the number of alveolar cent had electrocardiographic changes, which was 4 macrophages. Electron microscopic examination of times the expected rate for that age group. the lungs of rats that had inhaled particulate lead 3 Makasev and Krivdina424 observed a two-phase oxide (200 ug/m ) for 14 days revealed ultrastruc-

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tural damage (mitochondria and endoplasmic 2. Lead: Airborne Lead in Perspective. National Academy of reticulum) to the alveolar macrophages and the type Sciences, Washington, D. C. 1972. p. 166. I alveolar epithelial cells. Biochemically, a con­ 3. Muro, L. A. and R. A. Goyer. Chromosome damage in ex­ siderable loss in the activity ofthe benzopyrene hy- perimental lead poisoning. Arch. Pathol. £7:660-663, 1969. droxylating enzyme in the alveolar macrophages 4. Kanner, N. L. Functional state ofthe adrenal cortex under 433 was observed by Bruch et al. the influence of lead. Prob. Endo. Gormonther. //:82, Few studies have been made of the effects of lead 1965. (In Russian) Cited in: H. A. Waldron and D. Stofen. on the immunologic system in man. Reigart and Sub-clinical Lead Poisoning. Academic Press, Inc., New Graber434 studied 12 preschool children having ele­ York. 1974. 5. Geleriu, R. and H. Straus. Effects of lead administered vated free erythrocyte protoporphyrin and blood with goitrogenic agents (thiocyanates). In: Int'l Symp., En­ levels 2 40 figldl and seven nonlead-burdened vironmental Health Aspects of Lead. Amsterdam. 1972. p. children for evidence of impairment of their im­ 249-254. munological responses. They found no differences 6. Straus, H., R. Gekeriu, and C. lonut. Effect of lead on between the control group and the lead-exposed animals subject to preliminary and simultaneous treat­ ment of aeroionization. In: Int. Symp. Environmental group with reference to complement levels, to im­ Health Aspects of Lead. Amsterdam. 1972. p. 263-270. munoglobulins, or to anamnestic response to the 7. Minden, H., W. Zegarski, and P. Rothe. Fermentunter- tetanus toxoid antigen. suchungen bei experimenteller Bleivergiftung. Int. Arch. Hicks435 points out that there is a need for Gewerbepathol. Gewerbehyg. 20:461-470, 1964. Cited in: systematic epidemiological studies on the effects of H. A. Waldron and D. Stofen. Sub-clinical Lead Poison­ ing. New York, Academic Press, Inc. 1974. elevated lead levels on the incidence of infectious 8. Biondi, S. Intra-Leukocyte phosphatase in the peripheral diseases in man. The paucity of information cannot blood in sub-acute experimental lead poisoning. Folia support the formulation of any dose-response rela­ Med. 35:133-147, 1955. tionship at this time. 9. Rana, S. V. S. Histochemical approach to the toxic effects oflead on the activity of some enzymes in the kidney ofthe 11.12 THE GASTROINTESTINAL SYSTEM common ground squirrel funambulus pennanti. Folia Histochem. Cytochem. 72:193-196, 1974. Colic is usually a consistent early symptom of lead 10. Yagihara, T. Phosphatase activity in young rabbits ad­ poisoning, warning of much more serious effects that ministered lead. Ann. Paed. Japan. 6:489-494, I960. are likely to occur with continued and prolonged 11. Soldatovic, D. and C. Petrovic. Influence of lead on lead exposure. Although most commonly seen in in­ enzyme activity in animals poisoned by small amounts of dustrial exposure cases, colic is also a lead poisoning lead. Arch. Farm. 73:253-258. 1963. 12. Haeger-Aronsen, B. Experimental disturbance of symptom present in infants and young children. porphyrin metabolism and of liver catalase activity in 436 Beritic reported on the cases of 13 of 64 men guinea pigs and rabbits. Acta Pharm. Toxicol. 27:105-115, exposed on their jobs to occupational levels of lead. 1965. The 13 had colic, probably lead related, and con­ I 3. Sroczynski, J. and G. Yonderko. A picture ofthe proteins stipation. They had blood lead levels ranging from a of blood serum of rabbits during prolonged lead poison­ ing. Postepy Hig. Med. Dosw. 7 7:608-609, 1963. Cited in: little less than 40 to 80 ug/dl as determined by H. A. Waldron and D. Stofen. Sub-clinical Lead Poison­ polarography, a technique which tends to yield ing. Academic Press, Inc., New York. 1974. values lower than the actual blood levels. The diag­ 14. Belli, R., M. Maggio, and G. Arciello. Body temperature nosis of lead-caused colic was supported by findings during experimental poisoning with tetraethyl lead. Folia of high urinary coproporphyrin, excessive basophilic Med. 38:190-191, 1955. stippling, reticulocytosis, and some degree of 15. Apostolov, 1., Zapryanov, and V. Gylybova. Activity of lysomal enzymes in the blood serum of rats under experi­ anemia, all of which are other clinical signs of lead mental lead poisoning. Byull. Eksp. Biol. Med. poisoning. 52:1070-1971, 1976. Although these symptoms are well documented in 16. Innaccone, A., P. Boscolo, E. Bertoli, and G. Bombar­ the literature, there are insufficient data by which to dieri. In vitro effects of lead on enzymatic activities of rab­ bit kidney mitochondria. Experientia. 30:467-468, 1974. establish a dose-response relationship for an effect of lead on the gastrointestinal system. 17. Nathanson, J. A. and F. E. Bloom. Lead-induced inhibi­ tion of brain adenyl cyclase. Nature. 255:419-420, 1975. 18. Yamamoto, T., M. Yamaguchi, and H. Sato. Effect of cad­ 11.13 REFERENCES FOR CHAPTER 11 mium acetate on bone acid hydrolase activity in rats treat­ ed with lead acetate. Eisei Kagaku. 27:289-293, 1975. 1. Ulmer, D. D. and B. L. Vallee. Effects of lead on biochemical systems. In: Trace Substances in Environmen­ 19. Waldman, R. K. and E. K. Borman. A note on serum trans­ tal Health. II. D. D. Hemphill (ed.). Columbia, Missouri; aminase activity after lead absorption. Arch. Ind. Health. University of Missouri Press. 1969. p. 7-27. 79:431-433. 1959.

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Vopr. serum of patients with chronic industrial lead exposure. Med. Khim. 72:41-44, 1966. Int. Arch. Gewerbepath. Gewerbehyg. 20:11, 1953. Cited 40. Goyer, R. A. and R. Krall. Further observations on the in: H. A. Waldron and D. Stofen. Sub-clinical Lead morphology and biochemistry of mitochondria from kid­ Poisoning. Academic Press, Inc., New York. 1974. neys of normal and lead-intoxicated rats. Fed. Proc. 24. Hanke, J. Z. An attempt of evaluating the degree of 25:619A, 1969. poisoning endangering the organism on the basis of 41. Rhyne, B. C. and R. A. Goyer. Cytochrome content of enzymatic tests (alkaline and acid phosphatase, aldolase, kidney mitochondria in experimental lead poisoning. Exp. oxalate and pyruvic transaminase. Med. Pr. Mol. Pathol. 74:386-391, 1971. 74(3):223-238, 1963. Cited in: H. A. Waldron and D. 42. Walton, J. R. Granules containing lead in isolated Stofen. Sub-clinical Lead Poisoning. Academic Press, Inc., mitochondria. Nature. 243:100-101, 1973. 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