Measurement of Enzymes in Environmental Intoxications

MICHAEL M. LUBRAN, M.D., Ph.D.

U.C.L.A. School of Medicine, Department of Pathology, Harbor General Hospital Campus, Torrance, CA 90509

ABSTRACT The paper details the enzymes which have been shown to be affected by certain zenobiotics, principally industrial pollutants, toxic metals, toxic gases and food additives. The role of mixed function oxidases is discussed and methods of assessing their activity indicated.

One of the consequences of our indus been investigated experimentally in trial civilization has been the creation of a animals. Data obtained from humans dangerous environment for mankind. In have resulted mainly from accidental or dustrial wastes pollute the atmosphere occupational exposure to relatively high and find their way into our drinking wa concentrations, or prolonged exposure to ter; large-scale treatment of agricultural lower concentrations, of the chemicals. areas with herbicides and pesticides Few planned experiments have been creates health hazards for farm workers carried out. Damage to the liver, kidneys, and consumers of farm products; insec lungs, nervous system or other target or ticides may persist in the environment gans is usually easily detected; conven and be consumed by humans; photo tional laboratory tests, e.g., liver function chemical smog and motor-car exhaust tests, are adequate for their investigation. fumes contain toxic materials; toxic met However, chronic exposure to low con als, such as lead, mercury and manganese centrations of xenobiotics may not pro are released into the environment; food duce overt effects in man but may yet be additives, coloring materials and preser harmful, for example, by increasing the vatives may be hazardous. Nature, un risk of cancer (food additives) or aided by man, is responsible for some pulmonary and cardiovascular disease environmental hazards, such as ionizing (ozone, oxides of nitrogen, carbon radiation, local concentrations of radioac monoxide). tive isotopes, high concentrations of fluo Xenobiotics or their metabolites may rides and other toxic chemicals in rivers, accumulate in the body without produc as well as the sunshine necessary for ing detectable effects until a critical level smog production. is reached. It thus becomes important to Foreign substances found in the envi distinguish between body burden and ronment are called xenobiotics. This toxic concentrations of the chemicals. word has gained acceptance, although it Because of the wide differences in the really means ‘foreign living things’ and responses of experimental animals to more correctly describes extraterrestrial xenobiotics, it is unwise to extrapolate life forms. The toxicity of xenobiotics has from animal experiments to man, except in 210 MEASUREMENT OF ENZYMES IN ENVIRONMENTAL INTOXICATIONS 211 the most general terms. Information must rivative of the enzyme is rapidly hy be obtained directly by observation of drolyzed and the cholinesterase activity people exposed occupationally or other restored. Treatment is not required as the wise to xenobiotics. As enzymes are in patient recovers rapidly on being removed volved in the body’s responses to xenobi from the toxic agent. The phosphorylated otics, either in their metabolism or as a enzyme can be reactivated rapidly by consequence of their toxic action, changes treatment of the patient with certain in the activities of specific enzymes have oximes (e.g. pralidoxime chloride or been sought as indicators of toxicity. Un 2-PAMCL). Without treatment, the en fortunately, apart from enzymes associated zyme activity is restored at the rate of 1 to 2 with liver damage, there are few enzymes percent a day. specifically related to the toxic effects of Red cell AChE, being specific for individual xenobiotics. Possibly, the ones acetylcholine, is a better indicator of in described will be augmented by further hibition by organophosphorus pesticides study. than is serum PChE, which has a wider substrate specificity and may measure Cholinesterases non-specific esterase activity. However, a decrease in the activity of blood cholin These are esterases which hydrolyze esterase, whether in the red cell or in the choline esters more rapidly than other es serum, indicates only exposure to the pes ters.3, 16>20>28-37 Acetylcholinesterase ticide. There is no correlation between the (AChE), which is found in the red cell, is degree of enzyme inhibition and the clini most active towards acetylcholine, its nat cal state ofthe patient. The measured activ ural substrate. At least two AChE isoen ity depends on the analytical method used, zymes exist. Many cholinesterases (ChE) and the degree and duration of exposure of isoenzymes (11 or more) occur in serum, the worker. Treatment should be based on jointly being called pseudocholinesterase the clinical findings and the probability of (PChE). Pseudocholinesterase acts on a exposure to organophosphorus pesticides; variety of substrates, including acetyl laboratory investigations should be used choline. Cholinesterases are distin as confirmatory tests. The effect of pes guished from other esterases by being in ticides maybe heightened in subjects with hibited by 10 |U,M eserine. genetic abnormalities of ChE. Organo In addition, ChE are inhibited by or- phosphorus pesticides decrease the di- ganophosphorus compounds, carbamates bucaine and fluoride numbers as well as and some sulphonyl derivatives. Lipases cholinesterase activity. Some other and carboxylesterases are also inhibited by xenobiotics, e.g., ozone and oxides of ni these compounds, a phenomenon leading trogen, may decrease ChE activity. some investigators to infer falsely the exis tence of pancreatitis. Arylesterases, which can also hydrolyze choline esters, are not Enzymes and Toxic Metals inhibited by organophosphorus com pounds and are not included in the PChE Lead has been the most studied metal, group, although they may be active in but mercury, cadmium, and manganese some methods used for measuring PChE produce effects on enzyme activities. activity. Lead inhibits 8-aminolevulinic acid de Inhibition of cholinesterases is due to hydratase (ALAD), which catalyzes the the phosphorylation or carbamylation of condensation of two molecules of the active site. Phosphorylation is irre 8-aminolevulinic acid to porphobilino versible in the body, but the carbamyl de gen.14 Lead also inhibits ferrochelatase, 2 1 2 LUBRAN which is involved in the formation of pressed by exposure to lead, but not heme from iron and protoporphyrin. when normal blood was treated with Other enzymes concerned with heme lead.41 The diagnostic value of ALAD ac synthesis may also be affected. The tivity lies in its indication of lead expo logarithm of ALAD activity varies in sure; owing to its great sensitivity to lead, versely with blood lead concentration.36 it cannot be used as an index of lead in No inhibitory effect probably occurs at toxication. However, with the introduc concentrations below about 15 fig per dl, tion of methods in which inhibited and the value depending on the age of the uninhibited activities are measured, this subject.43 conclusion may be changed.14 Although there is ample evidence for Other red cell enzymes have been the inverse relationship between blood studied in subjects exposed to lead. lead concentration and ALAD activity, it Glutathione reductase activity increases is not clear that there is a strong correla as blood lead increases,15,33 possibly as a tion between ALAD activity and the toxic compensatory mechanism to overcome effects of lead, particularly when blood the reduction of SH groups brought about lead concentrations are not greatly ele by lead. Red cell glutathione decreases as vated.42,43 It may be possible that the ob lead increases. The activities of carbonic served inhibition by lead is an in vitro anhydrase isoenzymes B and C increase effect, as it is prevented by EDTA (which in subjects chronically exposed to low chelates lead) and is reversed in vitro by levels of lead.39 Catalase activity falls as the addition of thiol compounds such as blood lead rises.30 Recently, a new red glutathione and dithiothreitol; the en cell enzyme, 5'-ribonucleotide phos- zyme is strongly inhibited in vitro by the phohydrolase (E.C. 3.1.3.5) has been de addition of lead salts. scribed.24 It catalyzes the hydrolysis However, the toxic effects of lead in re of uridine - 5' - monophosphate and lation to ALAD activity have been asses cytidine-5'-monophosphate to uridine sed by the measurement in blood and and cytidine liberating orthophosphate. urine of heme precursors and derivatives The enzyme is very sensitive to lead, and by clinical evaluation of the subject. being almost completely inhibited by a These parameters are themselves insen 10~6 M concentration (about 20 fig per sitive to lead toxicity, except for erythro dl). In lead-exposed subjects, having cyte protoporphyrin (FEP). Maxfield and blood lead concentrations between 44 Henry21 concluded, on the basis of exper and 109 fig per dl, the enzyme activity iments in which blood from non-exposed was markedly depressed. Further studies dogs was mixed with blood from dogs ex are required to determine the value of posed to lead, that the observed lowered this enzyme as an indicator of lead tox ALAD activity in exposed dogs was due icity. to an in vivo effect. ALAD activity in the Although mercury combines with the liver is depressed in parallel with red cell SH groups of enzymes and modifies the ALAD in humans exposed to lead.36 activity of many enzymes in animal tis Further, the pH optimum for ALAD is sues,42 it has little measurable effect on different in lead-exposed subjects and blood enzymes in man, except for a de non-exposed subjects.21 Adding lead to creased activity of red cell carbonic normal blood depresses the ALAD activ anhydrase. Methyl mercury causes a ity but does not alter the optimum pH. slight decrease in ALAD activity.35 Similarly, ALAD could be reactivated by Cadium can substitute for zinc in some heat dénaturation when activity was de metalloenzymes; the effect on enzyme ac MEASUREMENT OF ENZYMES IN ENVIRONMENTAL INTOXICATIONS 2 1 3

tivity is variable.42 ALAD and glutathione the cases described previously) do not, as reductase activities are not affected, but a rule, produce measurable changes in red cell carbonic anhydrase activity and blood enzyme activity, unless organ serum plasmin activity rise.33 damage (usually the liver) occurs.22 Liver Manganese causes an increase in the enzymes are then elevated; occasionally, activity of serum adenosine deaminase in lipase9 activity is increased. However, severe and moderate cases of poison chronic exposure even to low levels of ing.10,23 many organic compounds produces de tectable changes in humans through their Enzymes and Toxic Gases effects on the mixed function oxidases of Exposure to oxides of nitrogen, ozone the liver, adrenal cortex and other organs, and carbon monoxide results in changes and hepatic drug metabolizing en in the activities of certain enzymes. Red zymes.6,12, 16,18,25,40 cell AChE is decreased and red cell and Mixed function oxidases (MFO) are in serum glutathione reductase increased in volved in the primary oxidation of or subjects exposed to 1 ppm of oxides of ganic compounds, usually producing OH nitrogen for two to three hours.7 Red cell groups. The resulting compounds are AChE and serum glutathione reductase then made more polar (water-soluble) by activities are decreased by exposure to conjugation with glutathione, glucuronic ozone (0.5 ppm for two to three hours);8 acid, sulphate, glycine and glutamine. Al the activities of red cell glucose-6-phos- though the oxidation products are usually phate dehydrogenase and lactate de less toxic than the original compounds hydrogenase increase. The clinical (detoxication), occasionally more toxic in significance of the enzyme changes is termediates are produced (toxication). not clear. For example, parathion is converted into Chronic exposure of experimental the toxic paraoxon. MFO’s are found in animals to low levels of carbon monoxide greatest concentration in the ribosomes causes polycythemia, increase in heart of the smooth endoplasmic reticulum; size and weight and an increase in the M high concentrations are also found in the component of the cardiac lactate mitochrondria of the adrenal cortex. dehydrogenase.26,27 The isoenzyme pat Many M FO’s, e.g., in the liver, use tern of humans exposed to carbon cytochrome P 450 for oxygen activation monoxide may show an increase of and nicotinamide-adenine dinucleotide M-containing isoenzymes.34 Carbon phosphate-reduced cytochrome c reduc monoxide had an effect on ALAD.4 The tase for restoring enzyme activity. A activity of this enzyme is lower in smok flavoprotein is used in the adrenal cortex. ers than in nonsmokers, there being an MFO’s and related enzymes are normal inverse relationship between log ALAD body enzymes, involved in enzymatic activity and carbon monoxide concentra degradation of endogenous steroids and tion (blood lead concentrations were not the detoxication and conjugation of waste measured). ALAD activity is not altered metabolites, utilizing pre-existing in vitro by saturating the blood with car biochemical pathways. Xenobiotics and bon monoxide. drugs having chemical structures making them suitable substrates for these en Mixed Function Oxidases and Drug zymes are metabolized by them. Many Metabolizing Enzymes xenobiotics, including food preserva Xenobiotics resulting from industrial tives, coloring agents, artificial food pollution of the environment (except in flavors and organic peroxides, can induce 214 LUBRAN hepatic microsomal enzymes; the phar subjects exposed to xenobiotics would macological effect of given amounts of reveal any effects they may have had on many drugs is affected.11 enzyme induction and would be of par Conversely, the enzyme inducing ef ticular value in foretelling the response fect of many drugs (particularly of the subject to certain classes of drugs. phenobarbitone) affects the metabolism of xenobiotics, which may be enhanced or depressed. As examples, polychlori References nated biphenyls (PCB) induce the hepa 1. Al v a r e s , A. P., F is c h b e in , A., Sa s s a , S., A n tic microsomal drug metabolizing en d e r s o n , K. 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