ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 7, No. 3 Copyright © 1977, Institute for Clinical Science 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