Mechanisms of chemical toxicity—a unifying hypothesis

REGULATORY

T~~I~~LOGY

Mechanisms

AND

PHARh4ACOLOGY

2,267-286

(1982)

of Chemical Toxicity-A

Unifying Hypothesis

DENNIS V. PARKE Department of Biochemistry, University of Surrey, Guildford, Surrey, United Kingdom

Received March I982

Most casesof chemical toxicity involve one or both of the fundamental pathological processes of “acute lethal injury” and “autoxidative cellular injury.” The process of “acute lethal injury” by which toxic chemicals interfere with cellular energy metabolism, leading ultimately to cell death and tissue necrosis, is well known and reasonably understood. Inhibition of glycolysis, mitochondrial respiration, or oxidative phosphorylation, resulting in lack of ATP synthesis or inhibition of ATPase and other enzymes, leads to decreased efficiency of the sodium pump, hydropic degeneration, lipid accumulation, and eventually cell death. Less well known are the mechanisms whereby toxic chemicals initiate autoxidation, leading to “autoxidative cellular injury,” disrupting cell membranes, and resulting in increased autophagocytosis, cell death, and mutations. Many reactive intermediates of toxic chemicals are electrophiles, free radicals, or free-radical generators, which may potentiate the toxicity of tissue oxygen, depleting intracellular glutathione and biological antioxidants, resulting in membrane damage, impairment of the calcium pump, cell death, and damage to DNA. The mechanisms of oxygen toxicity and chemical-mediated oxygen toxicity are discussed, with particular reference to the microsomal mixed-function oxidase system and its role in the detoxication and activation of environmental chemicals. The dependence of tissue oxygen concentration, the rates of oxidative activation of chemicals, and the extents of autoxidative cellular injury on the size of the animal species is considered, andthe importance of this to the scientific evaluation of chemical toxicity is discussed.

INTRODUCTION

The wide variety of molecular structures which are found in different toxic chemicals, and the diverse symptoms characteristic of their toxicities, suggest that chemicals may manifest toxicity by strictly individual mechanisms. Even if one selects a particular type of toxicity that is reasonably well defined and highly characteristic, such as carcinogenicity, a great diversity of chemical structures are seen to manifest this, e.g., polycyclic aromatic hydrocarbons, aliphatic nitroso compounds, mycotoxins, metals, and asbestos, so that no common mechanism of carcinogenicity would appear likely. Nevertheless, if this diversity of chemical structures is examined more closely, and the ultimate molecular mechanisms of toxicity studied, it is often possible to discern common mechanisms of toxicity. In carcinogenicity, for example, many organic compounds and metals, together with ultraviolet and ionising radiation, are known to be able to cause malignancies, but despite the diversity of these 267 0273-2300/82/040267-20$02.00/0 Chpyrigbt 0 1982 by Academic Prea, Inc. AU rig,bta of repmduction in any form reserved.

268

DENNIS V. PARKE CONJUGATION

BIOTRANSFORMATION (Phase

(Phase

1 metabolism)

2 metabolism) Conjugated

chemical

(proximate

Reactive lrltermediate (covalent binding, radical generation mutations)

carcinogen)

FIG. 1. Detoxication and activation of environmental chemicals by biotransformations tions.

and conjuga-

causative agents they have in common the ability to generate free radicals in vim, a phenomenon which is known to result in mutations and malignancy. Ultimately, toxicity is the result of chemical reaction, or reactions, of the toxic chemical, or its metabolites, with critical intracellular molecules. Chemicals which are electrophilic and highly reactive per se are generally too reactive to enter the cell unless present at high concentrations, but may be directly toxic, reacting especially with cell plasma membranes. However, most toxic chemicals require metabolic activation into reactive intermediates which are the ultimate intracellular toxic entities. Metabolism of environmental chemicals mostly occurs in mammalian liver and in similar tissues of other species, and either may result in deactivation of the chemical by processes of biotransformation and conjugation or, alternatively, may activate the chemical into more toxic intermediates (see Fig. 1). The choice of detoxication or activation depends on the nature of the chemical, the concentration Causes ACUTE

LETHAL

INJURY

Lack

Effects

of O2 and

Inhibition enzymes

nutrients

Defective

of glycolysis (metals, oxidants)

Inhibition of mitochondrial oxidative hosphorylation (CN , meta P s. barbiturates) Inhibition adenyl

of cyclases

ATP-ases and (detergents)

INJURY

Oxidants (quinones,

accumulation

Disorganisation of

Degranulation

mitochondria

lysosomes of E.R.

death,

Lipid

of Nat

of

Disruption

Cell

AUTOXIDATIVE

Nat pump

Cellular and water

tissue

necrosis

peroxidation

quinoneimines) Membrane

Free radicals (CC14, polycyclic Radical generators (nitroso compounds, quinones)

damage

hydrocarbons)Degranulation Defective

and Cat+

disruption

of E.R.

pump

semiDNA damage Malignancy,

FIG. 2. Fundamental mechanisms of toxicity.

and

mutations

cardiovascular

disease

GENETIC TOXICOLOGY/DRUG Exogenous .^

nutrients

269

SAFETY

Exogenous

nutrients .^

Impaired supply nutrients + Oz (ISCHAEMIA )

of

due to accumulation of water and electrolyte

1 Damaged , Mitocydrion ---.

ATP

formation

FIG. 3. Mechanisms of acute lethal injury. The diagrammatic representation shows the normal cell on the left and the damaged cell on the right. The damaged cell is turgid due to accumulation of water and electrolyte through disfunction of the sodium pump; mitochondria and lysosomes swell and disrupt.

present, the species of animal, its nutrition (Chow, 1979; Parke and Ioannides, Salocks, et al, (198 l), and many other biological factors. ACUTE

LETHAL

INJURY

AND AUTOXIDATIVE

CELLULAR

198 1;

INJURY

Apart from certain specific mechanisms of chemical toxicity, such as lethal synthesis by fluoroacetate and fluoroaliphatic acids and inhibition of acetyl cholinesterase by organophosphates, there are two basic mechanisms of chemical toxicity, namely, “acute lethal injury” and “autoxidative cellular injury.” Acute lethal injury has been widely studied and concerns the interference with intracellular energetics, resulting in the failure of the sodium pump of the cell, leading to accumulation of electrolytes and water in the cell, cell death, and tissue necrosis (La Via and Hill, 1975). It is fundamental to most mechanisms of toxicity, including poisoning by arsenic, phosphorus, cyanide, barbiturates, and paracetamol. Autoxidative cellular injury has been little studied, until recently, as the only methods available for detecting this phenomenon have been by morphological changes and by a relatively nonsensitive chemical method based on the determination of malondialdehyde formed during lipid peroxidation. The characteristics of these two fundamental mechanisms of chemical toxicity are shown in Fig. 2, and the mechanism of acute lethal injury is outlined diagramatically in Fig. 3. Immunological injury, characteristic of hyperimmune reactions, is mediated in its final stages by the mechanisms of autoxidative cellular injury.

270

DENNIS V. PARKE 1. Dctoxicotion mixed-

funct. -

Benzene

2.

Phenol

Phenylglucuronlde

Activation &J

c-oxyqenatio:

Radical

;&/g

H OH Benzdolpyreno-~S-ddhydrodid-9,10-epoxide

Benzobalpyrene

3.

excreted

format

ion

CCI,

-

cyt.P450

4. Ligand

complex

CF,CHClb

ccl;

formation

cyLP450 -

with

f-9

-

Cytochrome

CF,CH:

cyt. -

CHCI,+

OH’

P45O

P450 CF&H~Fe(P450)

FIG. 4. Metabolic fates of environmental chemicals in the endoplasmic reticulum.

THE ENDOPLASMIC MIXED-FUNCTION

RETICULUM OXIDATIONS

AND

The endoplasmic reticulum of a cell is concerned primarily with protein and glycoprotein synthesis, and in many tissues is also associated with the enzymes which detoxicate and activate environmental chemicals, including the mixed-function oxidase enzymes and certain conjugases, e.g., UDP-glucuronyl transferase. Many toxic chemicals are highly lipophilic and once they have entered the cell tend to accumulate in the lipid membranes of the endoplasmic reticulum. The endoplasmic reticulum metabolises these exogenous chemicals in a variety of ways including (1) detoxication by mixed-function oxidation and conjugation; (2) activation by C- and N-oxygenations, and conjugations; (3) formation of radicals by oxidative or reductive metabolism, and the initiation of free-radical chain reactions; (4) formation of carbenes, carbanions, and other reactive intermediates which form complexes with cytochrome P-450 and other components of the endoplasmic reticulum (see Fig. 4). Fundamental to these four types of reaction are the processes of microsomal mixed-function oxygenation, by which cytochrome P-450 and its reductase, present in the endoplasmic reticulum, can oxygenate carbon-, nitrogen-, and sulphur-contaming substrates with molecular oxygen (see Fig. 5). This cycle of cytochrome P45Odependent mixed-function oxygenation has various alternative pathways, including the generation of superoxy anion and hydrogen peroxide, both of which are

GENETIC TOXICOLOGY/DRUG

271

SAFETY

substmte-0 I

5. MicrosomaI mixed-function oxidation cycle.

FIG.

highly cytotoxic species. In addition, superoxy anion and possibly hydroxyl radicals can be generated directly by flavoprotein oxidases, by one-electron reductions by flavoproteins, such as cytochrome P-450 reductase, and by interaction of traces of inorganic iron with water in the presence of oxygen and traces of organic substrates. Superoxide anion may undergo further spontaneous reactions to generate peroxide, hydroxyl radical, and possibly singlet oxygen, all highly reactive, toxic species of oxygen (see Fig. 6) (Svingen, et al., 1979; Frank and Massaro, 1980; Halliwell, 198 1). Another microsomal oxygenase, prostaglandin endoperoxide synthetase (PES), contains two enzymic activities, and catalyses the oxygenation of polyunsaturated fatty acids (arachidonic acid) to the hydroperoxy endoperoxide (PGG2, by bisdioxygenation) and then to the hydroxy endoperoxide (PGH2, by the hydroperoxidase activity); prostacyclin synthetase converts PGH2 to the corresponding hydroxy ep oxide, prostacyclin (PGI2) (Mamett, 198 1; Ulhich et al., 198 1). Prostacyclin synthetase activity is due to a form of cytochrome P-450, found in the endoplasmic reticulum of the aorta, and, probably, thromboxane synthetase (found in blood platelets) is also a cytochrome P-450 enzyme (Ulhich et al., 1981). Prostaglandin synthetase (PES), however, is not a cytochrome P-450 enzyme (Egan et al., 198 I), but it can effect the co-oxygenation of many environmental chemicals, such as the oxygenation of benzo(a)pyrene 7,8-dihydrodiol to the corresponding 9, lo-epoxide (Sivarajah et al., 198 1; Mamett, 198 1). PES-dependent co-oxygenation may thus be important in the oxidation of environmental chemicals to toxic, mutagenic, or carcinogenic intermediates in those tissues that have low mixed-function oxidase

ADP-Fe3+ ADP-Fe2+

+ D2

20 2 *-+ H2°2 ADP-Fe2+

FIG.

+

,

cyt. P-450 reductase

ADP-Fe2+

3+ + 02'-

_____*

ADP-Fe

2tl+

w

H202

02*-

-

O2 + OH-+ OH'

+ H202

p

ADP-Fe3+

6. Mechanism of NADPHdependent

+ D2

+ OH-+

OH'

microsomal generation of active oxygen.

272

DENNIS

V. PARKE

activity (Marnett, 1981), such as lung (Boyd, 1980), breast (Greiner et al., 1980), and aorta (Bond et al., 1979) and may explain the organotropy of certain carcinogens and the association between environmental chemicals and cardiovascular disease (Bond et al., 1979). As release of polysaturated fatty acid is the rate-limiting step in hydroperoxide biosynthesis, metabolism of xenobiotics by the co-oxygenation pathway is likely to be initiated by events which trigger phospholipase activity and release of arachidonic acid (Marnett, 198 1). OXYGEN

TOXICITY

AND AUTOXIDATIVE

CELLULAR

INJURY

Intracellular oxygen has a high potential for toxicity because of the facility with which it is reduced to reactive species (superoxy anion, peroxide, hydroxyl radical) (Chance et al., 1979), and is thought to be responsible for the natural ageing of the biological system by repeated cellular damage (Harman and Eddy; Frank and Massaro, 1980), including mutations and malignancies (Totter, 1980). Indeed, antioxidants and free-radical scavengers, such as BHT (butylated hydroxyanisole) and ethoxyquin, increase animal life span (Clapp et al., 1979), decrease the lethality of ionizing radiation (Clapp and Satterheld, 1975), and inhibit the action of chemical carcinogens (Wattenberg, 1980). The smaller the living organism, the higher the basal rate of oxidative metabolism and the higher the oxygen tension within its tissues (Booth et al., 1967). The higher the tissue oxygen tension, the more rapid is the generation of reactive intermediates of toxic chemicals by mixed-function oxygenation, and the more rapid and extensive is the production of toxic oxygen free radicals (Frank and Massaro, 1980; Freeman and Crapo, 1981). Even in the most primitive living organisms, oxygen toxicity is a major problem, and has probably resulted in the development of the natural oxygen detoxicating system, biological antioxidants, free-radical scavengers, and redox buffer systems (glutathione) (Fridovich, 1978). The oxygen-detoxicating system comprises cytochrome P-450, superoxide dismutase, catalase, and glutathione peroxidase (Chance et al., 1979), by which intracellular molecular oxygen is progressively metabolised into superoxy anion, peroxide, and water. Cytochrome P-450 is ubiquitous in all living organisms, and Wickramsinghe and Villee (1975) have suggested that its primary role in single-cell organisms is the detoxication of oxygen. Without this oxygen-detoxicating mechanism, life of any form would be difficult, since the generation of free-radical forms of oxygen rapidly destroys biological macromolecules and biological membranes, resulting eventually in the death of the living organism. It is probably because of this that unicellular organisms have extremely short life spans, of only a few hours, even though they may be variously protected by cytochrome P-450, superoxide dismutase, catalase, and glutathione. A major biological development that helped overcome the cellular toxicity of oxygen was the evolution of multicellular organisms, with specialised intracellular, cellular, and tissue functions, which resulted in the overall lowering of intracellular oxygen tension. Consequently, the larger the multicellular organism, the lower the oxygen tension within the tissues, the lower the rate of oxidative tissue damage and mutations, and the longer the life expectancy. Nevertheless, even in the long-living species of mammalia, oxygen toxicity and oxidative tissue damage have been associated with various spontaneous degenerative

GENETIC

TOXICOLOGY/DRUG

SAFETY

273

disease states (Frank and Massaro, 1980), such as atherosclerosis (Parke, 198 l), rheumatoid arthritis (Greenwald, I98 l), cataract (Bhuyan et al., 198 l), and diabetes (Grankvist, 198 1). The initiation of lipid pet-oxidation is known to involve several different mechanisms (cytochrome P-450 reductase-, cytochrome P-450-, and Fe2+-mediated) but it is unlikely that the hydroxyl radical (OH) has a major role in physiological lipid peroxidation (Tien et al., 1981). In liver microsomal NADPH-dependent lipid peroxidation, the flavoprotein NADPH-cytochrome P-450 reductase is the initiator and is capable of generating hydroxyl radicals and forming malondialdehyde (Lai et al., 1979). This form of lipid peroxidation is independent of cytochrome P-450 (Baird, 1980). In cytochrome P-450-mediated lipid peroxidation, superoxide may be formed during the cyclic function of cytochrome P-450 (Estabrook et al., 1979). Cytochrome P-450 also has peroxidase activity and with various organic peroxides, such as cumene hydroperoxide, lipid peroxidation and destruction of cytochrome P-450 may occur, by a mechanism different from that stimulated by NADPH (Kulkami and Hodgson, 198 1). Liver nuclei may also produce substantial amounts of superoxide in the presence of NADPH, due to the flavoprotein FAD-monooxygenase (Patton et al., 1980). In NADPH/iron-dependent lipid peroxidation, free ferrous iron is essential and the role of NADPH is to maintain iron in the ferrous state (Kombrust and Mavis, 1980a). Superoxide dismutase, catalase, and hydroxyl radical scavengers have little inhibitory effect, so that oxygen free radicals are unlikely to be involved in the initiation of iron-dependent lipid peroxidation, and EDTA has no enhancing effect over free iron alone (Kombrust and Mavis, 1980a). However, the iron chelator, desferrioxamine, very effectively inhibits iron-catalyzed lipid peroxidation (Gutteridge et al., 1979) as does the iron-binding protein, lactoferrin (Gutteridge et al., 1981). Iron-EDTA catalyses the formation of hydroxyl radicals from peroxide, largely independent of superoxide (Winterboum, 1979), but hydroxyl radicals are not readily formed in biological fluids (Winterbourn, 1981). Peroxidation of microsomal lipids results in destruction of glucose 6-phosphatase, cytochrome P-450, but not cytochrome P-450 reductase; the toxic lipid peroxides may also cause tissue damage at distant sites (Hruszkewycz et al., 1978). Lipid peroxidation may be inhibited by antioxidants, such as vitamin E and ubiquinone (Galanopoulous, et al., 1982) and the highly effective 6-hydroxy- 1,Cdimethylcarbazole (Malvy et al., 1980), free-radical scavengers such as P-carotene and 1,6diazobicyclo(2,2,2)-octane (Packer et al., 1981), the hydroxyl radical scavengers such as dimethylurea (Fischer and Hamburger, 1980), and the superoxide radical scavengers such as ceruloplasmin (Goldstein et al., 1979). Certain mixtures of oxygen free radicals (OH plus H202, or OH plus O;- plus H202) are synergistic and result in greater biological damage than the individual radicals alone (Kong and Davison, 1980). Furthermore, at higher tissue oxygen tensions further superoxide is produced, and molecular oxygen interacts at the sites of damage to exacerbate the tissue injury (Kong and Davison, 1980). INITIATION OF AUTOXIDATIVE INJURY BY ENVIRONMENTAL

CELLULAR CHEMICALS

This spontaneous biological damage by oxygen toxicity may be greatly potentiated by toxic chemicals. Normally, the biological defenses against oxygen toxicity,

274

DENNIS V. PARKE TABLE

1

E~~~SOFCHLORINATEDHYDROCARBONSONRATLIVERCALCIUM

Time after administration of halocarbon (W 1 4 12 24

Calcium content &g/g liver) Control 42 40 43 41

+ f -t f

2 2 3 1

cc4 43i 3 52& 3 83 k 10 137 + 25

CC12CHz 401 47+115 f 122 -t

2 4 31 34

Phenobarbitoneinduced CHQ 44+ 6 56+ 5 105+ 6 216 f 30

Note. Control and phenobarbitone-treated rats were treated with corn oil, CCL, or CH2CC12 at a dose of 1 ml/kg; CHC& was administered at a dose of 0.3 mg/kg. Data are mean values + SEM of five samples (from Moore, 1980).

namely, superoxide dismutase, catalase, glutathione, etc., will adequately protect against moderate exposures to toxic chemicals, but do not so effectively protect against chemicals which initiate radical-generating chain reactions, e.g., lipid peroxidation is important in carbon tetrachloride poisoning but does not assume significance in bromobenzene poisoning until cellular glutathione is depleted (Smith et al., 1982). The effect of oxygen toxicity and of toxic chemicals may be increased by inhibition of the body’s defence systems, e.g., disulfiram and diethyldithiocarbamate increase oxygen lethality by inhibiting superoxide dismutase (Forman et al., 1980). Depletion of intracellular glutathione, by vinylidene chloride, phorone (diisopropylidene acetone), or diethylmaleate leads per se to increased lipid peroxidation, because of the central role of glutathione in the cellular defence system against toxic chemicals and autoxidation (Younes and Siegers, 1981). Furthermore, the antioxidant ethoxyquin fed to mice increases liver glutathione levels and protects against monocrotaline toxicity, whereas the antioxidants, vitamin C, vitamin E, and selenium, which do not increase liver glutathione, had no protective effect against monocrotaline (Miranda et al., 1981). Deficiency of selenium, together with glutathione deficiency, increases lipid peroxidation, indicating that the selenium-dependent glutathione peroxidase plays a major role in protecting the liver from peroxidative damage evoked by the cytochrome P-450 mixed-function oxidase system and other generators of active oxygen (Wendel and Feuerstein, 198 1). The glutathione protection against lipid peroxidation also involves a heat-labile, soluble, cytosolic factor of liver, which is not glutathione peroxidase, but may be glutathione transferase(s) (Burk et a/., 1980; McCay et al., 198 1). Pretreatment of animals with inducers of the microsomal mixed-function oxidase system markedly increases the potential of chemicals to initiate lipid peroxidation. The lipid peroxidation produced by a high dose of paracetamol is increased lo-fold by pretreatment with phenobarbitone and 20- to 30-fold by pretreatment with polycyclic hydrocarbons (Wendel and Feuerstein, 198 1). Although part of this increased activity may be due to the increased metabolism of the paracetamol to reactive intermediates, the increased activity and changed character of the cytochrome P450 mixed-function oxidase system is also likely to contribute to the enhanced lipid peroxidation.

GENETIC TOXICOLOGY/DRUG

275

SAFETY

FIG. 7. Generation of peroxide and the other active oxygen species by paraquat.

One of the characteristics of autoxidative and other damage to the endoplasmic reticulum is the failure of the microsomal calcium pump, leading to the accumulation of calcium ions within the cell (see Table 1). Failure of the microsomal calcium pump is one of the earliest events in the hepatotoxicity of many halogenated hydrocarbons and indicates that disturbance of calcium homeostasis may contribute to the chain of events leading to cell death (Moore, 1980; Lowrey et al., 198 1). This damage to the endoplasmic reticulum is accompanied by loss of the attached ribosomes (degranulation), inhibition of glycoprotein synthesis, and loss of many other vital functions. A further characteristic of autoxidative cellular injury produced by environmental chemicals is the marked increase in hepatic catalase activity, peroxisomal fatty acid oxidation (enoyl-CoA hydratase and palmitoyl-CoA oxidation), and proliferation of liver peroxisomes, seen following exposure of rats to acetylsalicyclic acid (Sakurai et al., 198 I), hypolipidaemic drugs (clofibrate), industrial plasticizers, or to vitamin E-deficient diets (Reddy et al., 1981). This would indicate increased production of peroxide in liver tissue, with lipid peroxidation and generation of free radicals, by a mechanism not yet fully defined. It has been seen that inorganic iron promotes autoxidation, producing destructive activated oxygen and free radicals (Kombrust and Mavis, 1980a). Biological systems are normally protected against this potent cytotoxic hazard by (i) the difficult absorption of inorganic iron; (ii) the tendency for inorganic iron to form nontoxic complexes, e.g., by ferritin, the macromolecular scavenger of iron; and (iii) biological Polyunsaturated fatty acids

Se Lipid alcohols

I i

\GSH J

Lipid L radicals

Lipid hydroperoxides

/ perox'dase

GSH

\‘SH

antioxidant5 (vit.CandEI

1

GSSG

dp

h

Lipid peroxidotion

/’ reductase

radical scavengers Membmne damaqe

\ NADPH L

NADP G-6-P dehydraqenase

\

/ Malondialdehyde

FIG. 8. Molecular mechanisms of autoxidative damage and the protective effects of glutathione.

276

DENNIS V. PARKE

FIG. 9. Generation of superoxy anion radicals by flavoprotein reduction of quinones.

antioxidant defence mechanisms. Iron-dependent autoxidation may damage carbohydrates, proteins, and DNA, as well as oxidise polyunsaturated fatty acids in lipid peroxidation (Gutteridge, 198 1); and the accumulation of inorganic iron in tissues and the depletion of cytochrome P-450 have been associated with rheumatoid disease (Parke, 1981; Blake et al., 1981). Depletion of hepatic cytochrome P-450 and increase of haem oxygenase are seen following poisoning with organotin compounds and with many other metals (Rosenberg et al., 1980). Paraquat. One of the most extensively studied chemicals that potentiates oxygen toxicity is the herbicide, paraquat. Paraquat is taken up selectively by the lung, where it is oxidised by microsomal enzymes to the free-radical form, which catalyses the reduction of oxygen to superoxy anion, with subsequent production of hydrogen peroxide and hydroxyl radicals (see Fig. 7). The overall autoxidative damage produced by paraquat involves lipid peroxidation with malondialdehyde formation, free-radical damage of membranes, and eventual cell death, although intracellular glutathione and antioxidants, such as vitamin E and vitamin C, tend to combat this (Boyd, 1980) (see Fig. 8). Aromatic compounds. Many aromatic compounds, e.g., benzene, are metabolised by mixed-function oxidation with the formation of catechols and quinols, which

OH

0

Oxidot ive 1 issue damage

radical generation

-

Free radical tissue

CiXdent -binding

FIG. 10. Molecular mechanisms of benzene toxicity.

darn+

GENETIC TOXICOLOGY/DRUG

SAFETY

277

subsequently undergo spontaneous oxidation to ortho- and paru-quinones. In a similar way, aromatic amines and nitro compounds are ultimately metabolised to ortho- and paru-quinoneimines. One-electron reduction of these quinones by flavoproteins, e.g., mitochondrial NADPH-ubiquinone oxidoreductase or cytochrome P-450 reductase, generates free-radical semiquinones, which subsequently interact with molecular oxygen to generate superoxy anion, and by a cycle of reactions function as free-radical generators, leading to cellular autoxidation, lipid peroxidation, and extensive tissue damage (Tunek et al., 1980; Powis et al., 1981) (see Fig. 9). The known radiomimetic toxicity and aplastic anaemia associated with benzene have been attributed to this mechanism of free-radical generation from the known metabolites, quinol and 1,2,4-trihydroxybenzene (Greenlee et al., 198 1) (see Fig. lo), and the autoxidation of quinol has been shown to interfere with intracellular microtubule function (Irons and Neptum, 1980). Similarly, the cytotoxicity of the anticancer antibiotics, adriamycin, daunorubicin, and mitomycin, is attributed to one-electron flavoprotein reduction (cytochrome P-450 reductase) to semiquinone free radicals which interact with molecular O2 to generate free radicals (superoxide, hydroxyl) that cause extensive DNA strand scission (Bachur et al., 1979; Berlin and Haseltine, 198 1). Halogenated hydrocarbons. Similarly, many halogenated hydrocarbons, including carbon tetrachloride, chloroform, and the anaesthetic halothane, are metabolised into free radicals through reductive dehalogenation by cytochrome P-450 (Ullrich et al., 1978), but the extensive lipid peroxidation, generation of malondialdehyde, and other tissue damage leading to hepatic necrosis are dependent on the presence of oxygen (Ahr et al., 1980). The autoxidative cellular injury may involve the formation of CC&Oi an oxygenated free radical of Ccl; (Tomasi et al., 1980) but, most probably, also involves the generation of superoxide and other free-radical species of oxygen (Ahr et al., 1980). The autoxidative effects of the activation of CCL, and other halocarbons to free radicals by cytochrome P-450 differ from those of Fedependent lipid peroxidation (Kornbrust and Mavis, 1980b). Halocarbon autoxidative injury is dependent on cytochrome P-450 and NADPH and causes loss of cytochrome P-450 (denatured to cytochrome P-420) more than peroxidation of polyunsaturated fatty acids (Kornbrust and Mavis, 1980b). Depletion of glutathione leads to lipid peroxidation (Anundi et al., 1980) and the toxicity of Ccl, (Harris and Anders, 1980) and CHC13 (Kluwe and Hook, 198 1) is potentiated by deficiency of GSH. Carbonyl chloride (COC&) is a major metabolite of CCL, (Ahr et al., 1980) and CHC13 (Pohl et al., 1980) and is detoxicated by interaction with GSH to form diglutathionyl dithiocarbonate (GSCOSG) (Pohl, et al., 198 1). As might be expected, free-radical scavengers, antioxidants, and the anti-peroxidative anti-inflammatory drug, tinoridine, protect against CCL, hepatotoxicity (Yasuda et al., 1980). In addition, the halocarbon free radicals may form carbanion complexes with the enzyme that formed them, cytochrome P-450 (Ahr et al., 1982), or may undergo further dehalogenation to form carbenes, which also subsequently form cytochrome P-450 ligand complexes (Ulhich et al., 1978; Ahr et al., 1980). These complexes of cytochrome P-450 do not exhibit the normal mixed-function oxidase activity. Instead, a microsomal hydroxylation characteristic of cytochrome P-448, or of a oneelectron flavoprotein oxidase, becomes manifest. A wide diversity of chemicals, (e.g., halothane, amphetamine, safrole) are metabolically activated by mixed-function oxidases to form reactive intermediates which subsequently combine to form ligand

278

DENNIS V. PARKE

600

-

.

r 500 t &

.

/

-

f! c5 400 s 300

-

2E 200 3

-

/ .

.

Pi

g

100

2.3

.

: 2.4

l -*

/

/,

2.5 Log htraperitoneal

I 26

2.7

Dose

FIG. Il. Ethane exhalation in paracetamol overdosage. The dose response of ethane evolution to paracetamol treatment in starved mice is shown. Each point represents the mean values of three animals (from Wendel et al. 1979).

complexes of cytochrome P-450, possibly with subsequent decoupling of the cytochrome from its reductase, and an enhanced generation of superoxy anion and hydroxyl radical. This may therefore constitute a major basic mechanism of chemical toxicity, characteristic of those chemicals which can be metabolised to form freeradical intermediates. Sulphur compounds. Carbon disulphide and thiocarbonyl (thiourea) and thiophosphonyl (parathion) compounds similarly undergo metabolism by cytochrome P-450, with loss of sulphur to form a sulphene-cytochrome P-450 complex that leads to loss of normal microsomal mixed function oxidase activity and production of superoxide with consequent destruction of cytochrome P-450 and autoxidative tissue injury (Obrebska et al., 1980; Torres et al., 1981). AUTOXIDATION-QUANTITATIVE

METHODOLOGY

The study of chemical-mediated biological autoxidation has been greatly hindered by the absence of suitable quantitative techniques. Autoxidation and lipid peroxidation may be measured by the lysis of red blood cells (Schulze and Kappus, 1980) and the formation of malondialdehyde (Asakawa and Matsushita, 1979), conjugated dienes (Sagai and Tappel, 1979), and other decomposition products of lipids, but more recently has been determined by the formation of alkanes, the ultimate products of free alkyl radicals generated by autoxidative processes (Burk and Lane, 1979; Sagai and Tappel, 1979; Frank et al., 1980). The abundance of these alkanes varies, and may be dependent on age (Sagai and Ichinose, 1980) and the type of autoxidation process involved; it is interesting that the normal alkane exhaled in human breath is isoprene (Gelmont et al., 198 1). Enhancement of biological autoxidation by toxic chemicals gives rise to a marked increase in the production of alkanes, even in the isolated perfused liver (Mtiller et al., 1981), isolated heptocytes (de Ruiter et al., 198 l), and isolated macrophages (de Ruiter et al., 1980) and in many instances shows a threshold and a dose response. This is illustrated by cases of overdosage with the drug paracetamol (Wendel et al.,

GENETIC TOXICOLOGY/DRUG

PI-PZNOLS one electron hydroryiation

SAFETY

279

EPOXIES CPOXidC w=

\ CONJUGATES

I OUINONES

GSH \ CONJUGATES

1 DlHYDRODlOLS

otisy L,,,qoxatJ/ AJUG ATEs FREE FIG.

RADICALS

DIOL-EPOXIDES

12. Alternative pathways of metabolic activation and detoxication of benzo(a)pyrene.

1979), which is known to be metabolised to a quinoneimine that may interact with flavoproteins and tissue oxygen to initiate a free-radical chain-generating system (de Vries, 198 1) (see Fig. 11). More recently, chemiluminescence has been proposed as a sensitive assay method for oxidative radical reactions (Boveris et al., 198 1). In a quantitative study of chemical-induced lipid peroxidation in isolated hepatocytes by various methods the sensitivity was found to decrease in the following order: ethane production > chemiluminescence > pentane production = malondialdehyde formation > fluorescent product formation (Smith et al., 1982). In rheumatoid arthritis, plasma levels of malondialdehyde have shown good correlation with the severity of the disease (Muus et al., 1979), thus further substantiating the association of this disease state with uncontrolled autoxidation, possibly Fe-mediated (Blake et al., 198 1; Parke, 198 1). , AUTOXIDATION

AND CHEMICAL

CARCINOGENESIS

It has recently been suggested that the mechanism of chemical carcinogenesis may also be dependent upon the generation of free radicals, as well as on the formation of DNA complexes (Totter, 1980). Many carcinogens are metabolised into quinone reactive intermediates, which themselves are highly mutagenic, but do not necessarily alkylate DNA directly (see Fig. 12). Stier (1980) has recently suggested that a fundamental mechanism of carcinogenicity of aromatic amines may be by metabolism into the corresponding nitroso compounds, which are free radicals and become stabilized by binding to intracellular molecules, thereby forming stabilized spin-trap radicals which, like quinones, can act as free-radical generators. This shows an interesting parallel to the mechanism, mentioned at the beginning of this paper, concerning the mode of action of ionising radiation in carcinogenesis. Further evidence that the reactive species of chemical carcinogens may be oxidants or free radicals is afforded by the inhibition of chemical carcinogenesis by antioxidants and free-radical scavengers (Wattenberg, 1980). Tumour promotion by phorbol esters is mediated at least in part by formation of active oxygen species generated in the tissue inflammatory response to the promoting agent (Witz et al., 1980). The products of lipid peroxidation are themselves mutagenic and probably car-

280

DENNIS V. PARKE 3-Melhylcholanthrene P4ea

Pknoburbllone P450

OH OH

Testosterone

7a-hydroxy

Br

16 a-hydroxy

Er

Bromobenzene OH

Benzolalpyrene

OH

FIG. 13. Alternative sites of microsomal mixed-function oxidation. Hydroxylation products formed by cytochrome P-448(induced by pretreatment of animals with 3-methylcholanthrene) are shown in the left-hand column; hydroxylation products formed by cytochrome P-450(induced by phenobarbitone) are shown in the right-hand column.

cinogenic, and malondialdehyde, a major end product of lipid peroxidation, has been shown to be mutagenic to Escherichia coli cells with an active DNA-repair system, inducing crossliking of DNA by chemical reaction with DNA bases (Yonei and Furui, 198 1). The frequency of chromosomal aberrations in cultured lymphocytes of patients with Fanconi’s anaemia (high risk of cancer and high frequency of spontaneous chromosomal aberrations) is positively related to oxygen tension, indicating that the site primarily affected by the Fanconi mutation concerns the integrity of the defence system against oxygen toxicity, and also confirms the relationship between oxygen and mutations (Joenje et al., 198 1). Many carcinogenic polycyclic aromatic hydrocarbons, for example benzo(a)pyrene and benzanthracene, are metabolically activated by the mixed-function oxygenases to form epoxides and epoxide diols. Recent studies have shown that those reactive intermediates which are mutagenic and capable of alkylating and damaging DNA contain oxygen in a sterically hindered or “bay-region” position (Parke and Ioannides, 1982). These sterically hindered positions of xenobiotics are not oxygenated by the normal hepatic cytochrome P-450, but appear to be substrates only for cytochrome P-448 or possibly one-electron flavoprotein oxidations (see Fig. 13) and the bay-region oxygenated products are not acceptable substrates for subsequent detoxication by enzymes such as epoxide hydrate (Levin et al., 1977). The “bay-

GENETIC TOXICOLOGY/DRUG

281

SAFETY

TABLE 2 INHIBITION

BY RETINOIDS

OF SUPEROXIDE PRODUCY~ION IN PHORBOL-STIMULATED POLYMORPHONUCLEAR LEUCOCYTES

Superoxide production (nmol/min)

Retinoid

Reduction of superoxide

in rate formation (S)

5.6

-

4.5 1.7

18 70

3.9

-

44 PM 270 PM

4.4 1.3

0 67

None Retinoic acid 48 /.tM 240 PM

3.6

-

2.4 1.9

33 47

None Retinol

50 PM 250 /.tM None Retinyl

HUMAN

acetate

Note. The cells (0.7 X lO“/ml) were suspended in balanced salt solution containing phorbol myristate acetate (71 rig/ml) and cytochrome c (0.57 m&ml). The rate of superoxide production was determined spectrophotometrically from the rate of cytochrome c reduction (from Witz et a/., 1980).

region” 9,1 O-oxygenation of benzo(a)pyrene 7,8-dihydrodiol to yield the ultimate mutagen/carcinogen, benzo(a)pyrene 7&dihydrodiol9, IO-epoxide, is catalysed by cytochrome P-448, and cytochrome P-450 is relatively unable to catalyse the formation of this reactive intermediate (Levin et al., 1977). Similarly, the activation of chemical carcinogens by N-oxygenation may be catalysed by cytochrome P-448 or by flavoproteins, but not by cytochrome P-450, and the activation of the highly mutagenic N-containing polyheterocyclic compounds present in tryptophan pyrolysates is also effected only by cytochrome P-448 (Nebert et al., 1979). Experimental tumour formation is known to be inhibited by retinoic acid and by retinoids (Parke and Ioannides, 198 l), and all-trans-retinol, retinyl acetate, and retinoic acid have been shown to inhibit superoxide anion radical production by human leucocytes stimulated with phorbol myristate acetate (see Table 2) (Witz et al., 1980). Similarly, a riboflavin-deficient diet fed to young rats resulted in increased lipid peroxidation, increased hepatic cytochrome P-450, but decreased cytochrome P-450 reductase, confirming the role of the flavoprotein reductase and of other flavoproteins in the generation of reactive oxygen species and free radicals (Taniguchi, 1980). SPECIES

DIFFERENCES

AND EXTRAPOLATION

OF DATA TO MAN

It is now well known that the smaller the animal species, the higher the rate of oxidative metabolism of environmental chemicals (Walker, 1980), probably because oxidative metabolism is oxygen dependent (Jones, 198 l), and tissue oxygen tension varies inversely with the body weight of the animal (Booth et al., 1967). As oxidative metabolism of chemicals is seen to be involved in their biological activation and the initiation of autoxidative reactions, and autoxidative damage is a function of

282

DENNIS

V. PARKE

tissue oxygen tension, the autoxidative cellular injury resulting from exposure to toxic chemicals will be much greater in the smaller animal species. Hence, even at equivalent doses, on a body weight basis, small experimental animals such as rat and mouse will generally exhibit much greater tendencies to experience autoxidative cellular injury, including mutations and malignancy, than will larger species such as man. These recent findings concerning oxygen toxicity, the dependence of chemical toxicity on metabolic activation by mixed-function oxygenation, and the potentiation of autoxidative cellular injury by toxic chemicals indicate that it may be more fundamental and more constructive to follow molecular events, such as the measurement of alkane production, as an index of chemical toxicity. Furthermore, in the hazard assessment of new chemicals, species differences in the rates of activation of chemicals by oxidative processes and in the rates of activation of oxygen and the generation of free radicals may have the most profound effects. Thus in the extrapolation of animal data to man these various molecular processes which mediate chemical toxicity must be carefully considered, precisely determined, and the species differences scientifically evaluated. The real risk to man must be very carefully assessed and there is increasing evidence that ignorance of, or disregard for, the scientific principles outlined in this paper, are resulting in toxicologists and regulatory bodies making erroneous estimates of the potential hazards to man of many chemicals. REFERENCES AHR, H. J., KING, L. J., NASTAINCZYK, W., AND ULLRICH, V. (1980). The mechanism of chloroform and carbon monoxide formation from carbon tetrachloride by microsomnl cytochrome P-450. B&hem. Pharmacol. 29,2855-286 1. AHR, H. J., KING, L. J., NASTAINCZYK, W., AND ULLRICH, V. (1982). The mechanism of reductive dehnlogenation of halothane by cytochrome P-450. Biochem. Pharmacol. 31, 383-390. ANUNDI, I., RAJS, J., AND H&BERG, J. (1980). Chloroacetamide hepatotoxicity: Hydropic degeneration and lipid peroxidation. Toxicol. Appl. Pharmacol. 55, 273-280. ASAKAWA, T., AND MATSUSHITA, S. (I 979). Thiobarbituric acid test for detecting lipid peroxides. Lipids 14,401-406.

BACHUR, N. R., GORDON, S. L., GEE, M. V., ANDKON, H. (1979). NADPH cytochrome P-450 reductase activation of quinone anticancer agents to free radicals. Proc. Nat. Acad. Sci. USA 76, 954-957. BAIRD, M. B. (1980). Microsomal NADPH-dependent lipid peroxidation does not require the presence of intact cytochrome P-450. Biochem. Biophys. Res. Commun. 95, 15 lo- 15 16. BERLIN, V., AND HASELTINE, W. A. (1981). Reduction of adriamycin to a semiquinone-free radical by cytochrome P-450 reductase produces DNA cleavage in a reaction mediated by molecular oxygen. J. Biol. Chem. 256,4747-4756. BHUYAN, K. C., BHUYAN, K. D., AND PODOS, S. M. (1981). Evidence of increased lipid peroxidation in cataracts. IRCS Med. Sci. 9, 126-127. BLAKE, D. R., HALL, N. D., BACON, P. A., DIEPPE, P. A., HALLIWELL, B., ANDGUTTERIDGE, J. M. C. (1981). The importance of iron in rheumatoid disease. Lancet 2, 1142-l 144. BOND, J. A., KOCAN, R. M., BENDI~T, E. P., AND JUCHAU, M. R. (1979). Metabolism of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene in cultured human fetal aortic smooth muscle cells. Life Sci. 25, 425-430.

BOOTH, J., BOYLAND, E., AND COOLING, C. (1967). The respiration of human liver tissue. Biochem. Pharmacol. 16,72 l-724. BOVERIS, A., CADENAS, E., AND CHANCE, B. (198 1). Ultra weak chemiluminescence: A sensitive assay for oxidative radical reactions. Fed. Proc. 40, 195-198. BOYD, M. R. (1980). Biochemical mechanisms in chemical-induced lung injury: Roles of metabolic activation. CRC Crit. Rev. Toxicol. 8, 103-176.

GENETIC TOXICOLGGY/DRUG

SAFETY

283

BURK, R. F., AND LANE, J. M. (1979). Ethane production and liver necrosis in rats after administration of drugs and other chemicals. Toxicol. Appl. Pharmacol. 50,467-478. BURK, R. F., TRUMBLE, M. J., ANDLAWRENCE, R. A. (1980). Rat hepatic cytosolic glutathione-dependent enzyme protection against lipid peroxidation in the NADPH-microsomal lipid peroxidation system. Biochim. Biophys. Acta 618, 35-4 1. CHANCE, B., SIES, H., AND BOVERIS, A. (1979). Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 527-605. CHOW, C. K. (1979). Nutritional influence on cellular antioxidant defense systems. Amer. J. Clin. Nutr. 32, 1066-1081. CLAPP, N. K., AND SATTERFIELD, L. C. (1975). Modification of radiation lethality by previous treatment with butylated hydroxytoluene. Radiat. Res. 64, 388-392. CLAPP, N. K., SATTERFIELD, L. C., AND BOWLES, N. D. (1979). Effects of the antioxidant, butylated hydroxytoluene (BHT) on mortality in BALB/c mice. J. Gerontol. 34, 497-50 1. DE RUITER, N., MULIAWAN, H., AND KAPPUS, H. (1980). Ethane production of mouse peritoneal macrophages as indication for lipid peroxidation and the effect of heavy metals. Toxicology 17, 265-268. DE RUITER, N., OTTENWALDER, H., MULIAWAN, H., AND KAPPUS, H. (198 1). Ethane formation of isolated rat hepatocytes due to carbon tetrachloride. Toxicol. Lett. 8, 265-27 1. DE VRIES, J. (198 1). Hepatotoxic metabolic activation of paracetamol and its derivatives phenacetin and benorilate: Oxygenation or electron transfer. B&hem. Pharmacol. 30, 399-402. EGAN, R. W., GALE, P. H., BAPTISTA, E. M., KENNICOTT, K. L., VAN DEN HEUVEL, W. J. A., WALKER, R. W., FAGERNESS,P. E., KEUHL, F. A., JR. (198 1). Oxidation reactions by prostaglandin cyclooxygenase-hydroperoxidase. J. Biol. Chem. 256, 7352-736 1. ESTABROOK, R. W., KAWANO, S., WERRINGLOER, J., KUTHAN, H., TSUJI, H., GRAF, H., ANDULLRICH, V. ( 1979). Oxycytochrome P-450: Its breakdown to superoxide for the formation of hydrogen peroxide. Acta Biol. Med. Germ. 38,423-434. FISCHER, L. J., AND HAMBURGER, S. A. (1980). Dimethylurea: A radical scavenger that protects isolated pancreatic islets from the effects of alloxan and dihydroxyfumarate exposure. Life Sci. 26, 1405-1409. FORMAN, H. J., YORK, J. L., AND RSHER, A. B. (1980). Mechanism for the potentiation of oxygen toxicity for disulfiram. J. Pharmacol. Exp. Ther. 212, 452-455. FRANK, H., HINTZE, T., BIMBOES, D., AND REMMER, H. (1980). Monitoring lipid peroxidation by breath analysis: Endogenous hydrocarbons and their metabolic elimination. Toxicol. Appl. Pharmacol. 56, 337-344.

FRANK, L., AND MASSARO, D. (1980). Oxygen toxicity. Amer. J. Med. 69, 117- 126. FREEMAN, B. A., AND CRAPO, J. D. (198 1). Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J. Biol. Chem. 256, 10986-10992. FRIDOVICH,I. (1978). The biology of oxygen radicals. Science 201, 875-880. GALANOPOULOUS, D. G., BOOTH, R. F. G., AND QUINN, P. J. (1982). Protection by ubiquinone against lipid peroxidation. Biochem. Sot. Trans. 10,20. GELMONT, D., STEIN, R. A., AND MEAD, J. F. (1981). Isoprene-The main hydrocarbon in human breath. Biochem. Biophys. Res. Commun. 99, 1456-1460. GOLDSTEIN, I. M., KAPLAN, H. B., EDELSON, H. S., AND WEISSMANN, G. (1979). Ceruloplasmin. A scavenger of superoxide anion radicals. J. Biol. Chem. 254, 4040-4045. GRANKVIST, K. (198 1). Alloxan-induced luminol luminescence as a tool for investigating mechanisms of radical-mediated diabetogenicity. Biochem. J. 200, 685-690. GREENLEE, W. F., SUN, J. D., AND BUS, J. S. (1981). A proposed mechanism of benzene toxicity: Formation of reactive intermediates from polyphenol metabolites. Toxicol. Appl. Pharmacol. 59, 187195. GREENWALD, R. A. (198 1). Effects of oxygen-derived free radicals on connective tissue macromolecules: Inhibition by copper-penicillamine complex, J. Rheumatol. 8, 9- 13. GREINER, J. W., MALAN-SHIBLEY, L. B., AND JANSS, D. H. (1980). Detection of aryl hydrocarbon hydroxylase activity in normal and neoplastic human breast epithelium. Life Sci. 26, 3 13-3 19. GUTTERILZE, J. M. C. (198 1). Thiobarbituric acid-reactivity foilowing iron-dependent free-radical damage to amino acids and carbohydrates. FEBS Lett. 128, 343-346. GUTTERIDGE, J. M. C., PATERSON, S. K., SEGAL, A. W., AND HALLIWELL, B. (198 1). Inhibition of lipid peroxidation by the iron-binding protein lactoferrin. B&hem. J. 199, 259-261. GUTTERIDGE, J. M. C., RICHMOND, R., AND HALLIWELL, B. (1979). Inhibition of the iron-catalysed formation of hydroxyl radicals from superoxide and of lipid peroxide by desferrioxamine. Biochem. J. 184.469-472.

284

DENNIS V.PARKE

HALLIWELL, B. ( 198 I). Free radicals, oxygen toxicity and aging. In “Age Pigments,” (R. S. Sohal, ed.). Elsevier, Amsterdam. HARMAN, D., AND EDDY, D. E. (1979). Free radical theory of aging: beneficial effect ofadding antioxidants to the maternal mouse diet on lifespan of offspring: Possible explanation of the sex difference in longevity. Age 2, lO9- 122. HARRIS, R. N., AND ANDERS, M. W. (1980). Effect of fasting, diethylmaleate, and alcohols on carbon tetrachloride-induced hepatotoxicity. Toxicol. Appl. Pharmacol. 56, 19 I- 198. HRUSZK~~YCZ, A. M., GLENDE, E. A., JR., AND RECKNAGEL, R. 0. (1978). Destruction of microsomal cytochrome P-450 and glucose 6-phosphatase by lipids extracted from peroxidized microsomes. Toxicol. Appl.

Pharmacol.

46, 695-702.

IRONS, R. D., AND NEPTUM, D. A. (1980). Effects of the principal hydroxy-metabolites of benzene on microtubule polymerization. Arch. Toxicol. 45, 297-305. JOENJE, H., ARWERT, F., ERIKSSON, A. W., DE KONING, H., AND OOSTRA, A. B. (1981). Oxygendependence of chromosomal aberrations in Fanconi’s anaemia. Nature (London) 290, 142-143. JONES,D. P. (1981). Hypoxia and drug metabolism. Biochem. Pkzrmuco2. 30, 1019-1023. KLUWE, W. M., AND HOOK, J. B. (1981). Potentiation of acute chloroform nephrotoxicity by the glutathione depletor diethyl maleate and protection by the microsomal enzyme inhibitor piperonyl butoxide. Toxicol. Appl. Pharmacol. 59, 457-466. KONG, S., AND DAVISON, A. J. (1980). The role of interactions between Oz, HaOz, *OH, e- and O;- in free radical damage to biological systems.Arch. Biochem. Biophys. 204, 18-29. KORNBRUST, D. J., AND MAVIS, R. D. (1980a). Microsomal lipid peroxidation. I. Characterization of the role of iron and NADPH. Mol. Pharmacol. 17, 400-407. KORNBRUST, D. J., AND MAVIS, R. D. (1980b). Microsomal lipid peroxidation. II. Stimulation by carbon tetrachlotide. Mol. Pharmacol. 17, 408-4 14. KULKARNI, A. P., AND HOLXXON, E. (1981). A comparison of NADPH and cumene hydroperoxidestimulated lipid peroxidation in mouse hepatic microsomes. Int. J. B&hem. 13, 8 11-8 16. LAI, C.-S., GROVER, T. A., AND PIETTE, L. H. (1979). Hydroxyl radical production in a purified NADPHcytochrome c (P-450) reductase system. Arch. Biochem. Biophys. 193, 373-378. LA VIA, M. F., AND HILL, R. B. (1975). “Principles of Pathobiology,” 2nd ed. Oxford Medical, New York. LEVIN, W., WOOD, A. W., Lu, A. Y. H., RYAN, D., WEST, S., CONNEY, A. H., THAKKER, D. R., YAGI, H., AND JERINA, D. M. (1977). Role of purified cytochrome P-448 and epoxide hydrase in the activation and detoxification of benzo(a)pyrene. In “ACS Symposium Series,” No. 44, “Drug Metabolism Concepts” (D. M. Jerina, ed.), pp. 99-126. Amer. Chem. Sot., Washington, D. C. LOWREY, K., GLENDE, E. A., JR., AND RECKNAGEL, R. 0. (1981). Rapid depression of rat liver microsomal calcium pump activity after administration of carbon tetrachloride or bromotrichloromethane and lack of effect after ethanol. Toxicol. Appl. Pharmacol. 59, 389-394. MALVY, C., PAOLETTI, C., SEARLE, A. J. F., AND WILLSON, R. L. (1980). Lipid peroxidation in liver: Hydroxy dimethyl carbazole a new potent inhibitor. Biochem. Biophys. Res. Commun. 95, 734-737. MARNETT, L. J. (198 1). Polycyclic aromatic hydrocarbon oxidation during prostaglandin biosynthesis. Lfe Sci. 29, 531-546. MCCAY, P. B., GIBSON, D. D., AND HORNBROOK, K. R. (1981). Glutathione-dependent inhibition of lipid peroxidation by a soluble, heat labile factor not glutathione peroxidase. Fed. Proc. 40, 199-205. MIRANDA, C. L., CARPENTER, H. M., CHEEKE, P. R., AND BUHLER, D. R. (198 1). Effect of ethoxyquin on the toxicity of the pyrrolizidine alkaloid monocrotaline and on hepatic drug metabolism in mice. Chem.-Biol.

Interact.

37, 95-107.

MOORE, L. (1980). Inhibition of liver-microsome calcium pump by in vivo administration of CCL,, CHCls and 1,l-dichloroethylene (vinylidene chloride). Reprinted with permission from B&hem. Pharmacol. 29, 2505-25 11, Pergamon Press, Ltd. MILLER, A., GRAF, P., WENDEL, A., AND SIES, H. (198 1). Ethane production by isolated perfused rat liver. FEBS Lett. 126, 24 l-243. Muus, P., BONTA, I. L., AND DEN OUDSTEN, S. A. (1979). Plasma levels of malondialdehyde, a product of cycle-oxygenasedependent and independent lipid peroxidation in rheumatoid arthritis: A correlation with disease activity. Prostaglandins & Med. 2, 63-65. NEBERT, D. W., BIGELOW, S. W., OKEY, A. B., YAHAGI, T., MORI, Y., NAGAO, M., AND SUGIMURA, T. ( 1979). Pyrolysis products from amino acids and protein: Highest mutagenicity requires cytochrome PI-450. Proc. Nat. Acad. Sci. USA 76, 5929-5933. OBREBSKA, M. J., KENTISH, P. A., AND PARKE, D. V. (1980). The effects of carbon disulphide on rat liver microsomal mixed-function oxidases, in vivo and in vitro. Biochem. J. 188, 107-l 12.

GENETIC

TOXICOLOGY/DRUG

SAFETY

285

PACKER, J. E., MANHOOD, J. S., MORA-ARELLANO, V. O., SLATER, T. F., WILLSON, R. L., AND WOLFENDEN, B. S. (I 98 1). Free radical and singlet oxygen scavengers: Reaction of a peroxy radical with &carotene, diphenyl furan and 1,4-diaxobicyclo(2,2,2)-octane. Biochem. Biophys. Res. Commun. 98, 90 I-906. PARKE, D. V. (198 1). The endoplasmic reticulum: Its role in physiological functions and pathological situations. In “Concepts in Drug Metabolism” (P. Jenner and B. Testa, eds.), Part B, pp. l-52. Dekker, New York. PARKE, D. V., AND IOANNIDES, C. (198 1). The role of nutrition in toxicology. Annu. Rev. Nutr. 1, 201234. PARKE, D. V., AND IOANNIDES, C. (1982). Role of mixed-function oxidases in the formation of biological reactive intermediates. In Biological Reactive Intermediates. II. Chemical Mechanisms and Biological Effects” (R. Snyder, D. J. Jollow, D. V. Parke, G. G. Gibson, J. J., Kocsis, and C. M. Wither, eds.). Plenum, New York. PATTON, S. E., ROSEN, G. M., AND RAUCKMAN, E. J. (1980). Superoxide production by purified hamster hepatic nuclei. Mol. Pharmacol. 18, 588-593. POHL, L. R., BRANCHFLOWER, R. V., HIGHET, R. J., MARTIN, J. L., NUNN, D. S., MONKS, I. J., GEORGE, J. W., AND HINSON, J. A. (1981). The formation of diglutathionyl dithiocarbonate as a metabolite of chloroform, bromotrichloromethane and carbon tetrachloride. Drug Metab. Dispos. 9, 334-339. POHL, L. R., MARTIN, J. L., ANDGEORGE, J. W. (1980). Mechanism ofmetabolic activation ofchloroform by rat liver microsomes. Biochem. Pharmacol. 29, 327 l-3276. POWIS, G., SVINGEN, B. A., AND APPEL, P. (1981). Quinone-stimulated superoxide formation by subcellular fractions, isolated bepatocytes, and other cells. Mol. Pharmacol. 20, 387-394. REDDY, J. K., LALWANI, N. D., DABHOLKAR, A. S., REDDY, M. K., AND QURESHI, S. A. (1981). Increased peroxisomal activity in the liver of vitamin E deficient rats. B&hem. Znternat. 3, 41-49. ROSENBERG,D. W., DRUMMOND, G. S., CORNISH, H. C., AND KAPPAS, A. (1980). Prolonged induction of hepatic haem oxygenase and decreases in cytocbrome P-450 content by organotin compounds. Biochem.

J. 190,465-468.

SAGAI, M., AND ICHINOSE, T. (1980). Age-related changes in lipid peroxidation as measured by ethane, ethylene, butane and permute in respired gases of rats. Z@ Sci. 27, 73 l-738. SAGAI, M., AND TAPPEL, A. L. (1979). Lipid peroxidation induced by some halomethanes as measured by in vivo pentane production in the rat. Toxicol. Appi. Pharmacol. 49, 283-291. SAKURAI, T., MIJAZAWA, S., OSUMI, T., FURUTA, S., AND HASHIMOTO, T. (198 I). Induction of peroxisomal @-oxidation by the administration of acetylsalicyclic acid. Toxicol. Appl. Pharmacol. 59, 8-14. SALOCKS, C. B., HSIEH, D. P. H., AND BYARD, J. L. (1981). Butylated hydroxytoluene pretreatment protects against cytotoxicity and reduces covalent binding of aflatoxin Br in primary hepatocyte culture. Toxicol. Appl. Pharmacol. 59, 33 l-345. SCHULZE, R. M., AND KAPPUS, H. (1980). Lysis of erythrocytes as a result of microsomal lipid peroxidation induced by Ccl, or FeCl2. Res. Commun. Chem. Pathol. Pharmacol. 27, 129-l 37. SIVARAJAH, K., LASKER, J. M., ANDELING, T. E. (198 1). Prostaglandin syntbetase-dependentcooxidation of (+)-benzo(a)pyrene-7,8dihydrodiol by human lung and other mammalian tissues. Cancer Res. 41, 1834-1839. SMITH, M. T., THOR, H., HARTZELL, P., AND ORRENIUS, S. (1982). The measurement of lipid peroxidation in isolated hepatocytes. Biochem. Pharmacol. 31, 19-26. STIER, A. (1980). Redox cycle of stable mixed nitroxides formed from carcinogenic aromatic amines. Xenobiotica 3, 67 I-680. SVINGEN, B. A., BUEGE, J. A., AND O’NEAL, F. 0. (1979). The mechanism of NADPH-dependent lipid peroxidation. The propagation of lipid peroxidation. J. Biol. Chem. 254, 5892-5899. TANIGUCHI, M. (1980). Effects of riboflavin deficiency on lipid peroxidation of rat liver microsomes. J. Nutr.

Sci. Vitaminol.

26, 401-413.

TIEN, M., SVINGEN, B. A., AND AUST, S. D. ( 198 1). Superoxide dependent lipid peroxidation. Fed. Proc. 40, 179-182. TOMASI, A., ALBANO, E., Lo=, K. A. K., ANDSLATER, T. F. (1980). Spin trapping of free radical products of CC& activation using pulse radiolysis and high energy radiation procedures. FEBS Lett. 122, 303306. TORRES, M., J~RVISALO, J., AND HAKIM, J. (1981). Effect of carbon disulfide on superoxide anion production by rat liver microsomes. Exp. Mol. Pathol. 34, 331-337. TOTTER, J. R. (1980). Spontaneous cancer and its possible relationship to oxygen metabolism. Proc. Natl.

Acad.

Sci. USA 77, 1763- 1767.

286

DENNIS

V. PARKE

TUNEK, A., PLATT, K. L., PRZYBYLSKI, M., AND OESCH, F. (1980). Multi-step metabolic activation of benzene, effect of superoxide dismutase on covalent binding to microsomal macromolecules, and identification of glutathione conjugates using high pressure liquid chromatography and field desorption mass spectrometry. Chem-Biol. Interact. 33, l-17. ULLRICH, V., CASTLE, L., AND WEBER, P. (198 1). Spectral evidence for the cytochrome P-450 nature of prostacyclin synthetase. Biochem. Pharmacol. 30, 2033-2036. ULLRICH, V., KING, L. J. WOLF, C. R., AND NASTAINCYZK, W. (1978). Carbenes and free radicals of haloalkanes as toxic intermediates. Advan. Pharmacol. Ther. 9, 13 1- 138. WALKER, C. H. (1980). Species variations in some hepatic microsomal enzymes that metabolize xenobiotics. Prog Drug Metab. 5, 1 l3- 164. WATTENBERG, L. W. (1980). Inhibition of chemical carcinogenesis by antioxidants. In “Carcinogenesis,” Vol. 5, “Modifiers of Chemical Carcinogenesis” (T. J. Slaga, ed.), pp. 85-98. Raven Press, New York. WENDEL, A., AND FEUERSTEIN, S. (1981). Drug-induced lipid peroxidation in mice. 1. Modulation by monooxygenase activity, glutathione and selenium status. Biochem. Pharmacol. 30, 25 13-2520. WENDEL, A., FEUERSTEIN, S., AND KONZ, K.-H. (1979). Acute paracetamol intoxication of starved mice leads to lipid peroxidation in vivo. Biochem. Pharmacol. 28, 205 I-2055. WICKRAMSINGHE, R. H., AND VILLEE, R. A. ( 1975). Early role during chemical evolution for cytochrome P-450 in oxygen detoxication. Nature (London) 256, 509-5 1 I. WINTERBOURN, C. C. (1979). Comparison of superoxide with other reducing agents in the biological production of hydroxyl radicals. Biochem. J. 182, 625-628. WINTERBOURN, C. C. (1981). Hydroxyl radical production in biological fluids. Roles of metal ions, ascorbate and superoxide. Biochem. J. 198, I25- 13 1. WITZ, G., GOLDSTEIN, B. D., AMORUSO, M., STONE, D. S., AND TROLL, W. (1980). Retinoid inhibition of superoxide anion radical production by human polymorphonuclear leukocytes stimulated with tumor promoters. B&hem. Biophys. Res. Commun. 97, 883-888. YASUDA, H., IZUMI, N., SHIMADA, O., KOBAYAKAWA, T., AND NAKANISHI, M. (1980). The protective effect of tinoridine against carbon tetrachloride hepatotoxicity. Toxicol. Appl. Pharmacol. 52,407-4 13. YONEI, S., AND FURUI, H. (198 I). Lethal and mutagenic effects of malondialdehyde a decomposition product of peroxidized lipids, on Escherichia coli with different DNA-repair capacities. Mut. Res. 88, 23-32.

YOUNES, M., AND SIEGERS, C.-P. (198 I). Mechanistic aspects of enhanced lipid peroxidation glutathione depletion in vivo. Chem.-Biol. Interactions 34, 257-266.

following