Environmental Health Perspectives

Some Studies and Comments on Hepatic and Extrahepatic Microsomal Toxication-Detoxication Systems. A Limited Discussion of Some of the Heterogeneities of These Systems and of Their Responses to Stimulation of "Induction" by James R. Fouts, Ph. D.*

Introduction and other purposes), or (such as chem- icals used in purifying the water or pollu- Much of what will be discussed in this tants that cannot be removed), or air (odors, paper can be referred to as drug , perfumes, and particulates-some added or drug-metabolizing systems and some fac- deliberately, some we can't or don't remove) tors which affect these systems, for example and which we ingest in the process of living by causing enzyme induction. If such terms and working in an environment increasingly as "" and "enzyme induc- filled with effectors of these enzyme sys- tion" are not further defined and explained, tems. To replace "drug metabolizing en- many people will still know what I am talk- zymes" with "xenobiotic or foreign chemical ing about and referring to. However, this metabolizing systems" recognizes part of the sloppy language may needlessly restrict the problem-some of the substrates of these interest and importance of some of the are not drugs; (b) drug or xeno- research going on in this field. Attempts to biotic metabolizing systems can cause acti- overcome this language-semantics barrier vation and inactivation of chemicals- have been partly successful and need to be sometimes even in sequence with or on the refined and extended. Some points needing same substrate. Thus, changes in activity of emphasis in my opinion are: (a) drug metab- the drug or xenobiotic metabolizing systems olizing systems have a number of substrates can either increase or decrease the that are not drugs. Some of these are of chemicals serving as substrates of these "normal" substrates such as steroids, fatty systems. Such changes can be specific for acids, vitamins, and hormones. Other sub- one substrate or apply to several substrates strates include chemicals that are present in and at the same time may make one sub- our food (such as preservatives, coloring strate more toxic and another less so. What agents, food additives for taste, odor control often determines toxicity and actions of a chemical is the balance between metabolism to toxic vs. less-toxic metabolites. This *Chief, Pharmacology and Toxicology Branch, National Institute of Environmental Health Sciences, balance may be shifted by increasing any or National Institutes of Health, Box 12233 Research all of these or by increasing Triangle Park, North Carolina, 27707 some more than others, as well as by the

October 1972 55 simpler examples of increasing one- kind throw up his hands in despair at this point (toxication) of reaction and decreasing since alternatives to "drug metabolizing another (detoxication) or vice versa. Thus, enzymes" are not easy to sell to anyone, let two points come out-drug or xenobiotic alone think up. But the lack of viable alter- metabolizing systems are not just "detoxi- natives has led to a lot of "clubs" who do cation mechanisms" and increases or induc- not communicate well with each other and tion of these systems can lead at the same the whole subject suffers. I will now close or different times to intoxication as well as this part of the introduction by choosing my detoxication relative to the "before induc- label for these systems: toxication- tion" stage; (c) toxication-detoxication sys- detoxication systems, only wishing I could tems include many reactions that are not somehow also add: ... that may use as oxidative in nature, are not cytochrome substrates drugs, foreign chemicals, or endog- P450 requiring, are not restricted to the enous lipid-class members such as steroids, , and can occur elsewhere than in the fatty acids, and vitamins, or endogenous microsomes. Therefore, to talk only about non-lipids like thyroxine, and certain amines hepatic microsomal drug oxidations and (even epinephrine or precursors), and may further to consider only those which involve even be systems that neither toxify nor is to talk about a very detoxify, but rather only metabolize these small (but vigorously and intensely studied) various substrates, and whose metabolism part of the body's systems for metabolizing can be characterized as oxidative and non- endogenous and exogenous chemicals. oxidative, synthetic and hydrolytic, involving One cannot really deal with these three many organs besides liver, and including a problem areas merely by recycling the lot of non-cytochrome P450 dependent nomenclature. Yet our use of convenient but systems. inexact items can, by repetition, cause some Having spent so much time on broadening dulling of our appreciation of the problems perspectives, I must now hasten to assure we are making for ourselves and colleagues the reader that I cannot possibly deal with in other disciplines who need to inform us all these matters in this paper. Indeed the and vice versa about common research main subjects covered will be few in number efforts with these systems. People who work and terribly selective since I prefer to write on "drug metabolizing enzyme systems" will about my own work and not that of others. tend to use drugs as substrates and think of This will not be a general review of drug or the systems primarily in terms of their roles xenobiotic metabolism, since I think plenty in therapeutics. Scientists who study "xeno- of these already exist. Instead, I wish to biotic metabolizing systems" will think write about three research projects that have largely in terms of non-drug substrates and been under study in my laboratory over the perhaps concentrate on the role of these last few years, and which I feel are of rele- systems and their interactions with model vance to the interface between "drug" substrates, industrial chemicals or environ- metabolism and environmental health science mental pollutants. Research on "steroid or as disciplines having identity problems that fatty acid metabolizing systems" will tend to are not dissimilar. These research projects I focus on the role of these enzymes in regu- wish to concentrate on are: (a) extrahepatic lating homonal functions and body homeo- toxication-detoxication systems; (b) age- stasis. Studies on "vitamin and non-steroid dependent changes in toxication-detoxication hormone metabolisms" will often concern systems in lung vs. liver, and (c) species- itself with nutritional balances, intermediary strain-individual differences in "induction" of metabolism and similar systems. The point hepatic toxication-detoxication systems by being that words do make a difference, the pollutants benzpyrene and DDT. labels do matter, and we ignore this human If the reader wishes a general review of weakness with some costs attached. One can "drug metabolism," or even specifically drug

56 Environmental Health Perspectives metabolism and its role in environmental tifying low levels of component or enzyme health, he should read elsewhere. Several activity especially in the presence of high very good reviews of the subject of "drug blanks due to things like blood - the liver metabolism" and selected aspects of the has high levels of most toxication- systems in liver, especially those requiring detoxication systems and contaminants or cytochrome P450 have appeared quite inhibitors of assays can often be removed recently. Some of these reviews are in book easily or at least lowered by perfusion, form; others are in journals or monographs. washing, etc.; (d) stability of systems - prob- Many conferences are being held on the lems in preparing components or enzyme subject and the conference proceedings often systems for assay before they are lost by appear very shortly thereafter. I cannot cytolysis or autolysis, problems in storage of begin to list all such "reviews" that have systems (freezing), problems in long-time been published even in the last 3 or 4 years. incubation especially with low enzyme activi- Inevitably, I would leave a key reference ties - the liver decomposes slowly, stores well, out, and probably that of a good friend. and is stable for most incubation or assay Having thus dusted off a few old griev- periods especially since content or enzyme ances and given you some idea of what I activity is so high; (e) quantity of tissue will say and will not even attempt to do, I available vs. quantity of tissue needed for can now turn to my subject with a some- assay, statistics, etc. - high activity and plenty what less guilty conscience. of homogeneous tissue makes liver almost ideal in experiments calling for repetitive sampling or biopsy, estimation of individual Extrahepatic Toxication-Detoxication Systems variability, purification of the enzyme The major research efforts in the field of systems, etc. "drug-metabolizing enzymes" have been con- Early studies in "drug metabolism" did cerned with systems in the liver. It is true occasionally include comparisons of liver that the liver of most animal species studied with other tissues, at least in the common is especially rich in enzyme systems that laboratory animals. From such studies it was metabolize chemicals of all kinds. It is also learned that most organs of excretion or true that most workers have been aware that exchange were likely to have at least some other body tissues had the ability to metab- capacity to metabolize chemicals. However, olize chemicals, but the reasons for the rel- it was also obvious from this early work that ative lack of studies of these other organs in most cases, the variety of toxication- are numerous and probably include: (a) ease detoxication systems was certainly much less of handling the tissue - isolating it for study in extrahepatic tissues (than in liver), their in vivo and in vitro as in perfused organ activities (or levels) were often barely quan- systems, slices, homogenates, and subcellular tifiable and likely to be highly variable in fractions - few tissues can match liver in "amount" from one species to another and manipulability for experimental study at all even one animal to another - seemingly these levels of organization; (b) hetero- much more so than with liver. From time to geneity of structure - irregularities of blood time publications have appeared dealing with flow or supply, lobular architecture or the metabolism of compound "X" by some similar organ area differences, number of extrahepatic tissue. Few if any studies have different cell types - the liver is almost attempted to characterize the "drug metabo- ideally homogeneous, having relatively uni- lizing systems" of extrahepatic tissues in form lobules, and only two or three major anything like the way done with liver. cell types which can now be separated from Among the problems that seemed to be each other for further study if desired; (c) rather consistently overlooked were: (a) ability to detect and quantify the metabo- Does the perfused whole organ metabolize lizing systems of interest - problems in quan- the drug or remove it from blood by bind-

October 1972 57 ing, storage or in some cases excretion?; (b) lung. We chose to study metabolizing sys- Do slices of the organ metabolize the drug tems in rabbit lung since this animal has (or store it, or excrete it)?; (c) What might high levels of several "drug metabolizing be the activity of drug-metabolizing enzymes enzymes" in its lung, and the mass of tissue in homogenates and subcellular fractions per animal made it more likely that we (mitochondria, nuclei, lysosomes, micro- could study systems in individual animals somes, soluble fraction) and how does this (1). There was also a system to study drug compare (per gram organ) with activity in metabolism in an isolated perfused lung (2). slices or in whole perfused organ?; (d) Have Our first studies were concerned with these subfractions of the homogenate been characterizing various treatments of lung to characterized by electron microscopy and obtain homogenates, and then with the iso- biochemical markers?; (e) How great a prob- lation from such homogenates of repre- lem is created by having many different cell sentative fractions such as nuclei, mito- types in the organ - which of these cell chondria, and microsomes. We hoped to types are active in drug metabolism?; (f) identify these subcellular fractions on the Have a variety of substrates been used, and basis of marker enzyme activities, using have enzyme assays been optimized with markers that were regarded as highly specific respect to protein dependence, substrate for the analogous hepatic fractions. concentration, pH and cofactor requirements, A major problem arises just in getting a ionic strength, etc.?; (g) What kinds of en- reasonably good homogenate of lung tissue. dogenous inhibitors or activators may be The compromise needed is between rupture present in vivo vs. in vitro and can these of at least a significant fraction of the cells inhibitors or activators be removed and iso- and extensive fragmentation of particulates lated for study?; (h) What are the species, like mitochondria. We obtained what we strain, sex, and individual variations seen in consider to be a reasonable approach to such whether these systems are present (or ab- a compromise by just mincing the tissue sent) and what is the variation in level of extensively with an ordinary meat grinder or activity of these extrahepatic systems in vivo tissue press, and then homogenizing in a and in vitro ? What is the role of genetics vs. teflon-glass Potter homogenizer. The homog- environment in the control of these vari- enate resulting from such manipulation can ances? then be subfractionated into nuclei, mito- Among tissues of obvious relevance to chondria, microsomes and soluble fractions, problems of environmental pharmacology though the correspondence of these lung and toxicology are those at the portals of fractions with analogously prepared liver entry or contact between ourselves and our fractions may not be too exact. There seems environment. Metabolism of chemicals at to be much less cell disruption in the case of such entrances or contact spots could be lung than liver and a greater degree of cross- most important to the action of these chem- contamination as seen both in electron icals - leading to activation or fixation of the micrographs of the fractions and in terms of chemical at its site of metabolism, or detoxi- marker enzymes. Especially interesting was cation and excretion of the chemical. the finding of a meshwork in the lung Among the first organs that we sought to microsomal fractions; this mesh seemed to characterize with respect to toxication- tie together several microsomal vesicles and detoxication systems, and to compare these ribosomal particles into a kind of clump. We with the analogous liver systems, was the believe the presence of such a meshwork will lung. We were aware of the fact that lung make study of lung microsomal subfractions had been shown to have some ability to especially difficult. Thus, liver microsomes metabolize chemicals and that animals can be subfractionated into several types seemed to differ widely with respect to rough-surfaced vs. smooth-surfaced (3-11) as levels of toxication-detoxication systems in well as further subdivisions of each of these

58 Environmental Health Perspectives (fractions of rough-surfaced membranes; somes were much lower than liver micro- magnesium binding vs. non-binding types somes; (d) many "drug metabolisms" were of smooth-surfaced membranes (12, 13)). also much less active in lung than liver These microsomal subfractions in liver can microsomes although some metabolisms were be relatively pure (by electron microscopy) equally active in microsomes from the two and contain differing amounts of drug- tissues of the rabbit. These results are found metabolizing enzymes. Similar studies in a series of publications from our labora- applied to the lung could be misleading tory (14-20). unless some way is found to break up the A number of other research projects have meshwork found in the microsomal frac- been stimulated by these studies. For one tions. At present, we are studying the use of thing the lung, unlike the liver is quite sonication to break up these meshworks and heterogeneous - having marked morphol- we find that sonication helps to increase the ogical differentiations (lobules, bronchi, yield of microsomes per gram liver and lung alveoli) as well as a plethora of different cell and the percentage of a microsomal marker types. These lung cell types are difficult to enzyme (benzphetamine demethylase) which isolate in pure or homogeneous populations. is recovered in the microsomal fraction of When we homogenize whole lung we are liver and lung. For example, of the total sampling a kind of average of all cells in the benzphetamine demethylase activity in the lung at the time of death. Since we found unsonicated homogenate, we find 60-80% of that lung microsomes from such a heter- this in the microsomal fractions prepared ogeneous sample did contain appreciable from liver homogenates but only 20-25% of levels of toxication-detoxication systems, it it in the microsomes from lung homogenate. became important to identify which cells When the homogenates are sonicated even had these systems and whether certain sys- briefly (10 secs-30 secs), the recoveries of tems might be found in only a few or even activity in microsomes are markedly in- one kind of cell. Several approaches to this creased at least in lung. Thus in liver, recov- problem are possible and we hope to use eries range from 80-100% of homogenate several of these: (a) histochemical - identify activity in microsomes while in lung from at least qualitatively and perhaps even semi- 50-75% of the homogenate activity is now quantitatively where certain of the present in microsomes (14). This suggests toxication-detoxication systems might be that sonication is a very useful adjunct to found by applying substrates whose metabo- routine homogenizing for purposes of in- lites can be fixed, stained and identified in creasing recovery of microsomes and con- tissue sections; (b) separation of cells by tents from tissue homogenates. The effects digestion of organ minces with collagenase of sonication were especially marked with hyaluronidase, etc., to liberate cells which lung and we believe this may result from may then be separated on the basis of size, effects on the microsomal meshwork. charge, shape, etc., using gels, electro- Our work with marker enzymes and sub- phoresis, centrifugation, etc.; (c) selective strates of the toxication-detoxication stimulation or inhibition of lung cell growth systems showed that lung and liver fractions - certain factors, diseases, chemicals or were similar in many respects. Many of the stresses applied to the animal may kill off a "drug-metabolizing" systems were concen- major cell type or cause its proliferation to trated in lung microsomes just as they are in markedly change its proportion of the total. liver. Major differences between lung and This might be combined with method (b) to liver seemed to be (a) glucose-6-phosphatase give even better separations for study, pro- was not a good marker for lung microsomes; viding the factor, disease, stress or chemical (b) glucuronyl transferase activity was not did not affect cells not stimulated or killed detectable in lung microsomes; (c) cyto- and did not cause losses of toxication- chrome b5 and P450 contents of lung micro- detoxication systems even in cells stimulated

October 1972 59 to proliferate; (d) growth of cells in culture Another finding of interest was that drug or use of lung tumors - this often results in metabolisms, cytochrome b5 and P450 levels favoring one cell type over another. and activities of NADPH-cytochrome c and Even before these studies are done, one or P450 reductases did not correlate well at the two simple experiments can be tried - for various ages in eigher lung or liver. It was example lung washouts (lavages) can give cell also obvious that at least in the lung a major populations especially rich in alveolar mac- problem would arise in trying to equate the rophages. When we subjected such cells to rate-limiting step of drug metabolism with analysis for toxication-detoxication systems the activity of NADPH-cytochrome c reduc- we found little if any detectable (20). Thus tase. Various laboratories have tried to at least one of the major cell types in lungs identify the key (rate-limiting) step in cyto- contributing to the lung microsomal fraction chrome P450-dependent drug metabolisms in is not a storehouse or site of concentration liver microsomes. At the present time, many of toxication-detoxication systems. workers believe that for mixed function oxidases (MFO) in these liver microsomes, Age-Dependent Changes in Toxication-Detoxication the most likely candidate for this key role is Systems in Lung vs. Liver NADPH-cytochrome P450 reductase (P450 The newborn of several animal species are reductase) activity. Thus rates of MFO- relatively deficient in the hepatic microsomal catalyzed detoxication-reactions in liver "drug metabolizing" enzyme systems. Such a microsomes do not correlate well with cyto- defect probably contributes to the relative chrome P450 or b5 levels, but do agree sensitivity of the newborn (compared to fairly well with P450 reductase levels. Even adults) to the actions and of a better correlation may be found if the P450 number of chemicals and drugs that are reductase activities are measured in the detoxified by these systems. I have been presence of substrates or other effectors of interested in the age-related changes in the systems, since P450 reductase activity is toxication-detoxication systems for many affected by substrates (29) and metal ions or years, with most of my studies dealing with ionic strength (30, 31). the liver systems (21-27). Having found lung At all ages studied, lung and liver micro- to contain appreciable amounts of some somes had very similar but age-dependent analogous metabolic systems (15-20), we levels of NADPH-cytochrome c reductase were interested in how these lung systems and where measurable, NADPH-cytochrome compared with liver during the first month P450 reductase. Our preliminary studies on of life. We were also hoping to see whether these enzyme activities and their response to different parts of some of the toxication- added ions or substrates suggest that the detoxication systems might develop at quite responses of these enzymes are also similar different rates in liver vs. lung, and to gain in the two organs, at least in the adult some more insight into whether the rate- rabbit. We have now studied several sub- limiting step for certain reactions might be strates of liver vs. lung microsomal different in liver vs. lung. toxication-detoxication systems. Two group- In the rabbit, we found that two general ings can be made: (a) substrates whose rate patterns of development of enzyme activity of metabolism per mg microsomal protein and cytochromes seemed to be present: (a) a per unit time is the same or greater in lung slow gradual increase in activity and level than in liver microsomes (benzphetamine during and beyond the first month of life - dealkylation, biphenyl hydroxylation, ethyl- typical of most of the systems in the lung demethylation, or p-chloro-N- and (b) a gradual increase to an age of about methylaniline demethylation), and; (b) sub- 2 weeks and then a sudden jump to adult strates or components whose metabolism or levels of activity - typical of most systems in level per mg protein is much less in lung the liver (14). than in liver microsomes (benzpyrene

60 Environmental Health Perspectives hydroxylase, aminopyrine demethylase, an- chemicals to microsomal suspensions in a iline hydroxylase, p-nitrobenzoic acid reduc- spectrophotometer cuvette) are affected, tase, and the cytochromes b5 and P450). If and (f) effects on the kinetic constants of activity is expressed per nmole P450 per the drug or chemical metabolisms. unit time then nearly all metabolisms in lung Two classes of hepatic drug-metabolizing are equal to (group b) or many times greater enzyme inducers are well-studied, and (group a) than those in liver. typical members of these classes are benz- If NADPH-cytochrome c reductase is the pyrene or 3-methylcholanthrene vs. pheno- same enzyme as NADPH-cytochrome P450 barbital or DDT. The enzyme induction by reductase (32, 33), and if NADPH- benzpyrene can be detected between 6 and cytochrome P450 reductase is the rate- 12 hours after animals are treated with benz- limiting step for MFO's dependent on pyrene, whereas stimulation by DDT is de- cytochrome P450, then it is not a simple tectable only much later; benzpyrene matter to understand how the rate-limiting stimulates only a few enzyme systems while steps in lung and liver can be at the same DDT causes increase in most of the level while substrate metabolisms such as toxication-detoxication systems in liver benzpyrene hydroxylase, aminopyrine microsomes; benzpyrene does not cause in- demethylase, and aniline hydroxylase differ crease in parenchymal cell smooth endo- by factors as great as five-fold in lung vs. plasmic reticulum, whereas DDT usually in- liver. The result is not qualitatively changed duces a massive proliferation of this system; whether activity is expressed per mg protein benzpyrene induces the formation of a spec- or per nmole of P450; the discrepancy is trally different P450 (P1450, P448) that can just as large. But now the question changes: be purified and shown to behave quite How can the rate-limiting step in lung be so differently from normal P450, whereas DDT much greater than in liver when the metab- appears to promote the formation only of olism of several chemicals (aminopyrine, more P450; substrate difference spectra after aniline, benzpyrene) occurs at the same rate benzpyrene seem to be increased only for in the two organs? those substrates whose metabolism is in- Species Differences in Induction of Hepatic creased and most other substrate spectra are decreased or qualitatively changed, whereas Toxication-Detoxication Systems DDT seems to cause only increases in spectra by the Pollutants, Benzpyrene and DDT if any change occurs at all; benzpyrene seems The hepatic "drug-metabolizing enzyme to decrease Km for those substrates whose systems" can be stimulated by a variety of Vmax is increased, whereas DDT affects only chemicals when these are ingested by the Vmax (per mg microsomal protein). In animal. The stimulation usually takes several reality, DDT probably does not affect even hours or days, and seems to involve syn- Vmax, since if expressed per nmole P450, thesis of more of these systems. Chemicals there is little change in this "constant" as which cause this hepatic enzyme "induc- would be expected if only more, but not tion" can be divided into several classes on different enzyme is being produced. the basis of (a) how soon the increase can Species and strain differences in response first be detected; (b) whether several micro- to inducers of hepatic microsomal drug somal systems or components are increased metabolism have been described, but are not or only a few; (c) whether the increase is widely recognized. Such variability in re- accompanied by a proliferation of the paren- sponse can be very large quantitatively, at chymal cell (smooth) endoplasmic reticulum; which point it becomes almost qualitative (d) whether cytochrome P450 increases or a (one species responding so poorly that for new cytochrome (P1 450, P448) is formed; practical purposes there has been no induc- (e) whether and how much substrate- tion). Such variability can be important in difference spectra (produced by adding explaining different chemical toxicities

October 1972 61 especially in tests of chronic toxicity, and in promazine sulfoxidation). Other pathways understanding interactions of chemicals or were increased in some strains, but not chemicals-drugs-environmental stresses, etc. others. Some of these differences may have Induction of toxication-detoxication systems been environmentally induced rather than can markedly shift such things as ED50 or determined by genetics, since the wild rab- LD50 or even qualitatively change chemical bits (cottontails, jack rabbits) obviously actions or toxicities. could have had quite different diets, etc., The basis for these variations in response both before and after delivery to our lab- to inducers of the hepatic enzyme systems oratory (wild rabbits in captivity do not eat, has been presumed to be both genetic and are highly stressed, etc.). environmental, and both have now been In later work, we discovered an apparent described, though not in great detail. difference in response of rats vs. mice to Whether and how much induction of the induction of hepatic toxication-detoxication hepatic systems occurs with any given in- systems by benzpyrene and DDT (35-41). ducer does indeed vary with diet (starvation Thus, at doses and schedules of dosing and vs. diets with abnormal fat content, protein at times after last dose of the inducer that content, or vitamin content), age of the clearly established hepatic microsomal en- animal, time of day (or light-eating cycles), zyme induction by DDT or benzpyrene in and presence or absence of other effectors rats and other species including monkeys of these systems (antibiotics, antimetab- (42), the mouse did not show such induc- olites, pesticides, disease, stress), and these tion or showed it only marginally (36). A factors can be lumped together (with many detailed study of the mouse liver systems others I have not listed) under the general failed to uncover any evidence for enzyme class of environmental influences. Genetic induction by benzpyrene (38, 39), although control of enzyme induction can be seen in we were able to show some enzyme induc- variations in response to inducers between tion by the related, but more potent poly- sexes (in rats) as well as between individuals, cyclic hydrocarbon 3-methylcholanthrene. strains, and species of animals. Others have reported that mice are less Several years ago, we showed that strains likely to respond to hepatic microsomal of rabbits differed in response of their he- enzyme inducers of the polycyclic hydro- patic microsomal enzymes to phenobarbital carbon class than are other species, espe- administration to the rabbit (34). The dif- cially rats (43). Most of these other findings ferences seen involved not only the gener- were made using 3-methylcholanthrene as ality of whether a given strain seemed to the inducer, not benzpyrene, and the accu- respond to phenobarbital or not and how mulated evidence began to suggest that we much, but whether a given hepatic drug were dealing with a quantitative, not a qual- metabolism responded in all or only a few itative difference - mice responded poorly to rabbit strains. Among the six strains of rab- an inducer that worked well in rats. bits, the California and English strains A very similar picture emerged with DDT. seemed to be generally less responsive to DDT did not affect most mouse liver micro- phenobarbital than the others, while Jack somal systems enough for the changes to be rabbits seemed to be more responsive to measured, whereas the related inducer, chlor- phenobarbital ("responsive" defined as total dane, could cause quantifiable responses in number of drug metabolisms significantly in- mice (35, 36). Again the accumulating evi- creased by phenobarbital compared with dence suggested a quantitative, not quali- total number of drug metabolisms studied). tative, difference in mouse vs. rat response As far as pathways were concerned, only one to the hepatic enzyme inducers, DDT and was induced in all strains (hexobarbital ox- benzpyrene. idase) while two were not increased in any However, work done in Dr. Nebert's lab strain (amphetamine deamination and chlor- (44, 45) suggested that mouse strains may

62 Environmental Health Perspectives differ qualitatively in their response to in- ments (41) would appear to show that duction of benzpyrene hydroxylase by poly- enzyme induction by benzpyrene in the cyclic hydrocarbons. These workers have Swiss Webster mouse does not require a shown that inducibility is strain specific and synthesis of P448 as is believed necessary by gene dependent as a simple autosomal dom- most people in the field. inant trait. Thus the C57BL/6N mouse One other point may perhaps need em- responded to inducers of benzpyrene hy- phasis before leaving this section. Although droxylase, while the DBA/2N mouse did we attempted to use the same strain of mice not. A most interesting result of these exper- in all our studies on DDT and benzpyrene as iments was that the "resistant" mouse inducers in mice vs. rats, we may not have (DBA/2N) was able to respond to other been able to do so. In the 7 years between classes of inducers such as phenobarbital. our first reported experiments (35) and our Also that resistance of benzpyrene hydrox- latest studies (40, 41), it is unlikely that the ylase to induction applied to extrahepatic Swiss Webster strain of mouse remained tissues as well as liver. Other polycyclic genetically unchanged. Thus, we may well hydrocarbons (than 3-MC) were effective or have started with a mouse genetically unable non-effective in the same way as 3-MC. to respond to either DDT or benzpyrene, Thus, none of these worked in any-tissue of and ended up with a mouse able to respond the DBA/2N mouse, though phenobarbital at least to high doses of these inducers. We could stimulate hepatic drug metabolisms in hope to continue our studies on genetic con- this mouse. trols of response to enzyme induction by Our most recent work with mouse liver benzpyrene or DDT in the same mouse responses to benzpyrene and DDT has used strains used by Nebert and coworkers (44, the same strain of mouse that we started 45). with at Iowa, i.e., mice which did not re- spond to benzpyrene, but did respond to Conclusion 3-methylcholanthrene and very slightly to DDT. In these latest studies we have greatly This paper has considered only a -few of increased the dose of inducer used and the many sources of heterogeneity in micro- shortened the period between last dose of somal toxication-detoxication systems and in inducer and the time of enzyme assay. We the response of these systems to effectors have now been able to show that this strain such as enzyme inducers. It should be of resistant mouse (Swiss-Webster) can re- obvious that this heterogeneity has many spond to benzpyrene and DDT as inducers bases including both genetic and environ- of hepatic microsomal enzymes and com- mental factors. Few of these have been ponents, but that the induction effect is sorted out or studied in any detail, and as very much less and much shorter in duration we find more of these, it will be increasingly than with rats (40, 41). A finding of interest difficult to isolate only one such effector for to us was that even in mice that showed a detailed study. Some of the newer technics measurable enzyme response to benzpyrene may help us to simplify our experiments so treatment, there was still no shift in the as to more clearly identify and study single cytochrome P450-CO spectra such as occurs influencing factors - e.g. - in vitro culture in other species (due to formation of P1 450 technics to study enzyme induction on or P448). Nebert and coworkers (44, 45) isolated homogeneous cell populations. It is have shown that mice responding to 3- unlikely that such simplified systems can methylcholanthrene treatment (the C57BL eliminate all multifactorial interactions, but strain) showed a shift from cytochrome the approach will be useful regardless. P450 to P448, while mice that didn't re- More important, in my opinion, is a wider spond (by enzyme induction) showed no recognition of the heterogeneity of these P450 to P448 transformation. Our experi- systems and their responses. Such recog-

October 1972 63 nition could hopefully discourage the gener- (from the National Institute of General alizations for which overeager problem- Medical Sciences, NIH #GM 12675) while solvers always grasp. It is obvious from the author was its Director. literally thousands of studies that toxication-detoxication systems are impor- REFERENCES tant determinants of the action and toxicity 1. Oppelt, W. W., et al. 1970. Comparison of of a wide variety of chemicals, drugs, pollu- Microsomal Drug Hydroxylation in Lung and Liver of Various Species. Res. Communs. Chem. tants, etc. However, it is also becoming Pathol. Pharmacol. 1: 43. obvious that whether and when these sys- 2. Rosenbloom, P. M. and Bass, A. D. 1970. A tems play key roles in determining chemical Lung Perfusion Preparation for the Study of effects, how they fluctuate and from what Drug Metabolism. J. Appl. Physiol. 29: 138. causes, can very much be almost whimsical 3. Fouts, J. R. 1961. The Metabolism of Drugs by Subfractions of Hepatic Microsomes. Biochem. in terms of the fragmentary knowledge we Biophys. Res. Commun. 6: 373. now possess. Better understanding will hope- 4. Fouts, J. R., Rogers, L. A. and Gram, T. E. fully generate some more general principles 1966. The Metabolism of Drugs by Hepatic than we now possess. My purpose in writing Microsomal Enzymes. Studies on the Intra- this paper was to emphasize how few gener- microsomal Distribution of Enzymes and Rela- tionships Between Enzyme Activity and Struc- alities we now have and some of the areas ture of the Hepatic Endoplasmic Reticulum. that we have chosen to try to understand Exp. Mol. Pathol. 5: 475. better. 5. Gram, T. E., Rogers, L. A. and Fouts, J. R. 1967. Further Studies on the Metabolism of Drugs by Subfractions of Hepatic Microsomes. Acknowledgements J. Pharmacol. Exp. Ther. 155: 479. 6. Gram, T. E. and Fouts, J. R. 1967. Effect of Work by the author and colleagues as Pretreatment of Rabbits with Phenobarbital or reported in this paper has been done at the 3-Methycholanthrene on the Distribution of Drug-Metabolizing Enzyme Activity in Subfrac- University of Iowa, Department of Pharma- tions of Hepatic Microsomes. J. Pharmacol. cology, College of Medicine; the Oakdale Exp. Ther. 157: 435. Toxicology Center, Department of Pharma- 7. Gram, T. E. and Fouts, J. R. 1967. Further cology, the University of Iowa at Oakdale, Studies on the Metabolism of Drugs by Subfrac- Iowa; and the Pharmacology and Toxicology tions of Hepatic Microsomes. III. Effects of the NADPH-Generating System. J. Pharmacol. Exp. Branch of the National Institute of Environ- Ther. 158: 317. mental Health Sciences, National Institutes 8. Gram, T. E., Hansen, A. R. and Fouts, J. R. of Health, Research Triangle Park, North 1968. The Submicrosomal Distribution of He- Carolina. The author should acknowledge patic UDP-Glucuronyl Transferases in the the contributions of his former students Rabbit. Biochem. J. 106: 587. 9. Fouts, J. R. and Gram, T. E. 1969. The Metab- (graduate students, medical students, post- olism of Drugs by Subfractions of Hepatic doctoral students, clinical pharmacology Microsomes. The Case for Microsomal Hetero- trainees, foreign exchange visitors), and col- geneity. In: J. R. Gillette et al. (eds.) Micro- leagues (including technicians) to the work somes and Drug Oxidations. Academic Press, N. reported herein. Such a list of important Y. 81. 10. Fouts, J. R. 1971. Liver Smooth Endoplasmic contributors to this work would include Reticulum "Microsomal" Drug Metabolizing most of the authors cited in the reference Enzyme System. In: A. Schwartz, (ed.) Meth- list as attached, and I hope the reader will ods in Pharmacology. Appleton-Century-Crofts, use that bibliography in lieu of a listing of N.Y. 287. those names in this place. The research done 11. Dallner, G. 1963. Studies on the Structural and Enzymic Organization of the Membranous Ele- at the University of Iowa was supported by ments of Liver Microsomes. Acta Path. Micro- numerous research grants from the American biol. Scand., Suppl. 166. Cancer Society and the National Institutes 12. Dallman, P. R., et al. 1969. Heterogeneous of Health, but especially the grant sup- Distribution of- Enzymes in Submicrosomal porting the Oakdale Toxicology Center Membrane Fragments. J. Cell Biol. 41: 357.

64 Environmental Health Perspectives 13. Glaumann, H. and Dallner, G. 1970. Subfrac- and Liver During the First Month of Life. tionation of Smooth Microsomes from Rat Toxicol. Appl. Pharmacol. Liver. J. Cell Biol. 47: 34. 29. Gigon, P. L., Gram, T. E. and Gillette, J. R. 14. Fouts, J. R. and Devereux, T. R. in press. Devel- 1969. Studies on the Rate of Reduction of opmental Aspects of Hepatic and Extrahepatic Hepatic Microsomal Cytochrome P450 by Drug Metabolizing Enzyme Systems: Micro- Reduced Nicotinamide Adenine Dinucleotide somal Enzymes and Components in Rabbit Phosphate: Effect of Drug Substrates. Mol. Liver and Lung During the First Month of Life. Pharmacol. 5: 109. J. Pharmacol. Exptl. Ther. 30. Peters, M. A. and Fouts, J. R. 1970. A Study 15. Bend, J. R., Hook, G. E. R. and Gram, T. E. of Some Possible Mechanisms by Which Mag- 1971. Study of the Mixed-Function Oxidase nesium Activates Hepatic Microsomal Drug System in Rabbit Lung. Fed. Proc. 30: 282. Metabolism In Vitro. J. Pharmacol. Exp. Ther. 16. Bend, J. R. et al. 1971. Mixed-Function Oxi- 173: 233. dase (MFO) Activity of Microsomal Fractions 31. Fouts, J. R. and Pohl, R. J. 1971. Further from Rabbit Lung and Liver. The Pharm- Studies on the Effects of Metal Ions on Rat acologist 13: 245. Liver Microsomal Reduced Nicotinamide 17. Hook, G. E. R., et al. 1971. Comparative Dis- Adenine Dinucleotide Phosphate - Cytochrome tribution of Particulate Marker Enzymes in P450 Reductase. J. Pharmacol. Exp. Ther. 179: Rabbit Liver and Lung. The Pharmacologist 13: 91. 245. 32. Masters, B. S. S. et al. 1971. Immuno- 18. Bend, J. R., et al. (In Press). A Comparative chemical Studies on Electron Transport Chains Study of the Hepatic and Pulmonary Micro- Involving Cytochrome P450. I. Effects of Anti- somal Mixed-Function Oxidase Systems in the bodies to Pig Liver Microsomal Reduced Rabbit. J. Pharmacol. Exp. Ther. Triphosphopyridine Nucleotide - Cytochrome c 19. Hook, C0. E. R., et al. (In Press). Preparation of Reductase and the Non-heme Iron Protein from Lung Microsomes and a Comparison of the Dis- Bovine Adrenocortical Mitochondria. J. Biol. tribution of Enzymes Between Subcellular Frac- Chem. 246: 4143. tions of Rabbit Lung and Liver. J. Pharmacol. 33. Glazer, R. I., Schenkman, J. B. and Sartorelli, Exp. Ther. A. C. 1971. Immunochemical Studies on 20. Hook, G. E. R., Bend, J. R. and Fouts, J. R. the Role of Reduced Nicotinamide Adenine in press. Mixed-Function Oxidases and the Alve- Dinucleotide Phosphate - Cytochrome c (P450) olar Macrophage. (Biochem. Pharmacol). Reductase in Drug Oxidation. Mol. Pharmacol. 21. Fouts, J. R. and Adamson, R. H. 1959. Drug 7: 683. Metabolism in the Newborn Rabbit. Science. 34. Cram, R. L., Juchau, M. R. and Fouts, J. R. 129: 897. 1965. Differences in Hepatic Drug Metabolism 22. Hart, L. G., et al. 1962. Stimulation of Hepatic in Various Rabbit Strains Before and After Pre- Microsomal Drug Metabolism in the Newborn treatment with Phenobarbital. Procs. Soc. Exp. and Fetal Rabbit. J. Pharmacol. Exp. Ther. Biol. Med. 119: 1045. 137: 103. 35. Hart, L. G. and Fouts, J. R. 1965. Further 23. Fouts, J. R. and Hart, L. G. 1965. Hepatic Studies on the Stimulation of Hepatic Micro- Drug Metabolism During the Perinatal Period. somal Drug Metabolizing Enzymes by DDT and Ann. N. Y. Acad. Sci. 123: 245. Its Analogs. Arch. Exp. Path. Pharmak. 249: 24. Fouts, J. R. 1965. The Metabolism of Drugs by 486. the Foetus. In: J. M. Robson et al. (eds.). 36. Cram, R. L. and Fouts, J. R. 1967. The Influ- Embryopathic Activity of Drugs. J. A. Church- ence of DDT and y-Chlordane on the Metab- ill, Ltd., London. 43. olism of Hexobarbital and -Zoxozolamine in 25. Fouts, J. R. 1968. Hepatic Microsomal Drug Two Mouse Strains. Biochem. Pharmacol. 16: Metabolism in the Perinatal Period. In: K. 1001. Adamsons (ed.). Diagnosis and Treatment 37. Hansen, A. R. and Fouts, J. R. 1968. Influence of Fetal Disorders. Springer Verlag, N.Y. of 3,4-Benzpyrene or y-Chlordane on the Rate 291. of Metabolism and Acute Toxicity of Amino- 26. Eling, T. E., et al. 1970. Diphenylhydantoin pyrine, Hexobarbital and Zox4zolamine in the Effect on Neonatal and Adult Rat Hepatic Drug Mouse. Toxicol. Appl. Pharmacol. 13: 212. Metabolism. J. Pharmacol. Exp. Ther. 171: 127. 38. Hansen, A. R. and Fouts, J. R. 1971. A Com- 27. Eling, T. E., et al. 1970. Kinetic Changes in parison of the Effects of Benzpyrene Admin- Microsomal Drug Metabolism with Age and istration on Some Hepatic Microsomal Mixed- Diphenylhydantoin Treatment. Eur. J. Phar- Function Oxidases of Rats and Mice. Biochem. macol. 11: 101. Pharmacol. 20: 3125. 28. Fouts, J. R. and Devereux, T. R. (In press). 39. Hansen, A. R. and Fouts, J. R. (In press). Some Microsomal Enzyme Systems in Rabbit Lung Problems in Michaelis-Menten Kinetic Analysis

October 1972 65 of Benzpyrene Hydroxylase in Hepatic Micro- DDT. Gastroenterology 51: 213. somes from Polycyclic Hydrocarbon-Pretreated 43. Alvares, A. P., Schilling, G. and Levin, W. 1970. Animals. Chemico-Biol. Interactions. Species Differences in the Induction of Micro- 40. Chhabra, R. S. and Fouts, J. R. (In press). somal Hemoproteins and 3,4-Benzpyrene Induction of Hepatic Microsomal Drug Metabo- Hydroxylase by Phenobarbital and 3- lizing Enzymes in a Strain of Mouse Previously Methylcholanthrene. J. Pharmacol. Exp. Ther. Believed to be Resistant to DDT or 3,4- 175: 4. Benzpyrene (BP) Treatment. The Pharma- 44. Gielen, J. E., Goujon, F. M. and Nebert, D. W. cologist. 1972. Genetic Regulation of Aryl Hydrocarbon 41. Chhabra, R. S. and Fouts, J. R. (In press). Hydroxylase Induction. II. Simple Mendelian Stimulation of Hepatic Microsomal Drug Metab- Expression in Mouse Tissues In Vivo. J. Biol. olizing Enzymes in Mice by DDT and 3,4- Chem. 247: 1125. Benzpyrene. Toxicol. Appl. Pharmacol. 45. Nebert, D. W., Goujon, F.M. and Gielen, J. E. 42. Juchau, M. R., Gram, T. E. and Fouts, J. R. 1972. Aryl Hydrocarbon Hydroxylase Induction 1966. Stimulation of Hepatic Microsomal Drug- by Polycyclic Hydrocarbons: Simple Autosomal Metabolizing Enzyme Systems in Primates by Trait in the Mouse. Nature 236: 107.

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