Clofibrate pretreatment in mice confers resistance against hepatic lipid peroxidation.

Pretreatment with peroxisome proliferators protects mice against various hepatotoxicants. Since our previous work suggested that the hepatoprotection may involve an increased ability to cope with oxidative stress, the present work directly addressed this possibility. Several observations indicated a heightened defense against oxidative stress accompanies the hepatoprotection produced by clofibrate. Firstly, the carbonyl content of hepatic proteins from clofibrate-pretreated mice was 40% lower than those from vehicle-treated controls. Secondly, liver homogenates from clofibrate-pretreated mice produced less thiobarbituric acid reactive substances upon incubation under aerobic conditions or exposure to ferrous sulfate. This effect was not due to lower levels of peroxidation-prone polyunsaturated fatty acids in clofibrate-treated livers. Thirdly, in vitro experiments indicated that the antioxidant factor in liver homogenates from clofibrate-pretreated mice was not glutathione. Rather, since it was inactivated by proteases and heat treatment, we concluded that a protein is involved. Collectively, our results suggest that a resistance to lipid peroxidation develops in mouse liver during exposure to clofibrate. The identity of the putative antioxidant protein and its contribution to the protection against liver toxicity observed in this and other laboratories awaits future investigation.

Clofibrate-induced in vitro hepatoprotection against acetaminophen is not due to altered glutathione homeostasis.

Prior induction of peroxisome proliferation protects mice against the in vivo hepatotoxicity of acetaminophen and various other bioactivation-dependent toxicants. The mechanisms underlying such chemoresistance are poorly understood, although they have been suggested to involve alterations in glutathione homeostasis. To clarify the role of glutathione in this phenomenon, we isolated hepatocytes from mice in which hepatic peroxisome proliferation had been induced with clofibrate. The cells were incubated with a range of acetaminophen concentrations and the extent of cell killing after up to 8 h was assessed by measuring lactate dehydrogenase leakage from the cells. Hepatocytes from clofibrate-pretreated mice were much less susceptible to acetaminophen than cells from vehicle-treated controls. However, the extent of glutathione depletion during exposure to acetaminophen was similar in both cell types, as were rates of excretion of the product of glutathione-mediated detoxication of acetaminophen's quinoneimine metabolite, 3-glutathionyl-acetaminophen. The glutathione-replenishing ability of clofibrate-pretreated cells after a brief exposure to diethyl maleate also resembled that of control cells. More importantly, prior depletion of glutathione by diethyl maleate did not abolish the resistance of clofibrate-pretreated cells to acetaminophen. Taken together, these findings indicate that although glutathione-dependent pathways may contribute to hepatoprotection during peroxisome proliferation, the resistance phenomenon is not due exclusively to this mechanism.

Carcinogen risk assessment. Can we harmonise?

Harmonisation of risk assessment (RA) is one of the priorities for sound chemical management set by Chapter 19, Agenda 21 of the United Nations Conference on the Environment and Development (UNCED) 1992 Earth Summit. The benefits of harmonisation are self evident and include transportability and consistency of RA outcomes, transparency and efficiency of process, and credible science. The outcomes of carcinogen RA are a description or classification of the carcinogenic hazard, the conditions under which cancers may be induced, and an estimate of a dose or exposure which poses a minimal, or otherwise defined, risk in exposed human populations. Weight-of-evidence based systems which classify carcinogenic hazards are part of, but do not substitute for, the risk assessment process. Carcinogen RA is based on assessment of appropriate toxicological and exposure data sets, which may have much in common. However, national policy frameworks can differ to the extent that RA outcomes may be quite different for the same chemical(s). Historically, differences in science policy have been greater for cancer RA compared to other toxic endpoints, with a tendency to differentiate cancer RA on the basis of presumed mechanism (i.e. genotoxic or non-genotoxic) and relevance to humans (some carcinogenic responses in animals may be considered not relevant for human RA). Significant strides towards harmonisation are being made, with reassessment of some national policies and participation in international harmonisation programmes, such as the ones being managed by the International Programme for Chemical Safety (IPCS). Alternative approaches to quantitative carcinogen RA are being considered which are more amenable to harmonisation, and one such approach being developed in Australia in connection with contaminated sites will be discussed.

Effect of occupational exposure to aldrin on urinary D-glucaric acid, plasma dieldrin, and lymphocyte sister chromatid exchange.

The effects of exposure to the chlorinated cyclodiene termiticide aldrin was evaluated in pest control workers potentially exposed to this material. Sister chromatid exchange (SCE) frequencies were not elevated in workers handling aldrin. This is consistent with the fact that chlorinated cyclodienes are not genotoxic. Plasma dieldrin concentrations (up to 250 ng/ml) confirmed exposure in workers actively performing termiticide treatments and in maintenance and store workers, when compared with unexposed control workers (median concentration, 4.8 ng/ml). Urinary D-glucaric acid (DGA), an index of hepatic enzyme activity, was elevated in pesticide-exposed groups but urinary DGA was poorly correlated with plasma dieldrin level. This indicates that concurrent exposures of these groups to other pesticides may have influenced mixed-function oxidase metabolic activity.

Sister chromatid exchanges in lymphocytes of petroleum retailers.

Occupational exposure to petroleum vapour was assessed in workers employed in suburban petroleum retail outlets. Urinary output of thioethers provided a non-specific estimate of exposure to chemicals metabolised via a mercapturic acid pathway. Urinary d-glucaric acid (DGA) excretion was taken as an estimate of hepatic enzyme activity. Sister chromatid exchange frequency in lymphocytes was used as an indicator of genotoxic response to exposure. Workers were classified according to their employment at self service (where customers operate petrol pumps) or at driveway attended service stations (at which an employee operates the pumps), and according to exposure to cigarette smoke on the basis of urinary cotinine excretion. Prework and postwork urine samples of workers employed at driveway attended petrol stations contained more thioether than did those of self serve workers. When classified according to smoking behaviour there were no statistically significant differences, although thioether excretion tended to be higher in smokers than in nonsmokers. Urinary DGA excretion was similar in the two exposure groups. Cigarette smokers excreted more DGA, however, than nonsmokers. Sister chromatid exchange frequencies were higher in driveway attendants than in self serve personnel. When the influence of cigarette smoking was investigated there was a significant increase of sister chromatid exchange with combined exposure to petrol and cigarette smoking, but not with either factor alone. Correlation analysis showed that urinary cotinine concentrations were positively associated with urinary excretion of thioether and DGA, indicating that cigarette smoke induces the activity of hepatic enzymes and acts as a source of substrates metabolised through a thioether pathway. In conclusion it seems that exposure to petroleum vapour causes increased sister chromatid exchange in circulating lymphocytes of cigarette smokers, possibly as a result of enhanced hepatic conversion of vapour components to reactive metabolites. Urinary thioether output does not clearly discriminate between workers exposed to different amounts of petroleum vapour at retail outlets.

Biological and biological-effect monitoring of workers exposed to 4,4'-methylene-bis(2-chloroaniline).

4,4'-Methylene-bis(2-chloroaniline) (MOCA), a curing agent used in polyurethane manufacture, is a genotoxic and carcinogenic amine. This study aimed to assess occupational exposure to MOCA using as indices: (1) the post-work urinary output of MOCA; (2) urinary thioethers, assuming that conjugation with glutathione might be a significant pathway for the elimination of putative electrophilic metabolites of MOCA; and (3) sister-chromatid exchange (SCE) frequency in peripheral lymphocytes as an indicator of genetic damage. Process workers at a polyurethane production unit were found to have up to 38 mumol MOCA mol-1 creatinine in their urine at the end of a work shift. Smaller quantities were found in the urine of laboratory and supervisory staff, but none was detected in the urine of a group of office and sales staff from an unrelated industry, who served as unexposed controls. There was no evidence of MOCA-related urinary thioether output. There was a graded increase in SCE frequency from controls to process workers, consistent with their apparent exposure to MOCA. Administration of MOCA to rats (5 daily i.p. injections of 125 or 250 mg kg-1 resulted in dose-related increases in MOCA excretion and in lymphocyte SCE frequency, but there was no change in thioether output. These results indicate that urinary thioether excretion is inappropriate for monitoring MOCA exposure, but that where MOCA exposure can be demonstrated, by the presence of MOCA in urine, this is associated with genetic damage in both man and in the rat.

Species differences in the genotoxicity of cyclophosphamide and styrene in three in vivo assays.

Species differences in dispositional factors such as distribution, metabolism and excretion may often account for species differences in the toxic responses to foreign chemicals. In this study we compared the genotoxic responses of cyclophosphamide (CP) and styrene (ST) between Porton rats and LACA Swiss mice in three in vivo assays (bone marrow micronucleus (MN), sperm morphology (SM) and sister-chromatid exchange (SCE) assays). The sensitivities of the three assays were compared by the doses of the compounds required to elicit a significant genotoxic response. The baseline levels for the MN, SCE and SM assays were 1.1-1.4 and 1.2-1.3 MNPCEs/1000 PCEs, 0.23-0.24 and 0.20-0.21 SCEs/chromosome, 3.5-5.7% and 1.6-1.9% abnormal sperm in mice and rats, respectively. CP was a potent genotoxin in the MN and SCE assays but weakly genotoxic in the SM assay. At comparable doses, the rat was approximately 3-, 2.5- and 1.8-fold more sensitive to CP than mice in the MN, SM and SCE assays, respectively. ST produced weak genotoxic responses in all assays in mice and only in the SM and SCE assays in rats. The mice were more sensitive to ST in the MN and SM assays, while it was difficult to compare the species in the SCE assay. For both compounds the sensitivity of the three assays, in decreasing order, were SCE greater than MN much greater than SM. For CP the relative responses in the Porton rats and LACA Swiss mice were qualitatively similar to previous reports. Although the use of different strains may explain differences between the studies in the magnitude of the responses observed. The results for ST in the rat shows that the choice of genotoxic endpoint can determine whether a response is detectable. Moreover, the discrepancies between the results for ST in this study and others, suggest that as well as using a battery of in vivo tests, it may be prudent to select more that one strain or species to fully assess a compound's ability to produce DNA damage.

Influence of vegetable oil vehicles on bone-marrow proliferation in the mouse micronucleus test.

The detection of genotoxins in the mouse bone-marrow micronucleus (MN) test is sensitive to factors which may inhibit bone-marrow proliferation. We have shown that three commercially available cooking oils (olive, peanut and sunflower seed oils), commonly used as vehicles in toxicological tests, were able to induce a cytotoxic effect in mouse bone marrow. The effects observed were reversible and the magnitude of the responses varied with the oil administered. The results suggest a need to examine vehicle effects when conducting the MN test.

Implication of alterations in intracellular calcium ion homoeostasis in the advent of paracetamol-induced cytotoxicity in primary mouse hepatocyte monolayer cultures.

We have examined the fluctuation of free cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) using the fluorescent probe quin-2 during the cytotoxic response induced by low concentrations (100-250 mum) of the model hepatotoxin paracetamol (APAP) in primary mouse hepatocyte cultures over 5 days. APAP-associated increases in [Ca(2+)](i) were recorded prior to APAP-associated cytotoxicity, and correlated with the subsequent loss of cell viability as measured by intracellular lactate dehydrogenase and K(+) efflux. Co-incubation with promethazine (1 mum) or ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic 0215 acid (4 mm) attenuated both the APAP-associated [Ca(2+)](i) changes and cytotoxicity. These results support the hypothesis that mobilization of intracellular Ca(2+) may be an important early event in APAP-induced hepatotoxicity.

Induction of microsomal drug metabolism in man and in the rat by exposure to petroleum.

To determine the effect of petroleum exposure on the activity of hepatic mixed function oxidase enzymes, salivary elimination kinetics of antipyrine were determined in 19 petrol station attendants and compared with 19 controls. Antipyrine half life in petrol station attendants was shorter than in controls. Microsomal preparations (10 000 x g supernatants) were prepared from six male Porton rats exposed to petrol vapour (5 ppm at an air flow rate of 41/min for eight hours a day for three weeks) and six control rats maintained under the same conditions without exposure to petrol vapour. The rates of oxidative metabolism of antipyrine, aminopyrine, ethylmorphine, aniline, and benzo(a)pyrene were all increased by more than 45% in the petrol-exposed rats. The results indicate that petrol vapour is a moderately potent inducer of mixed function oxidase activity in rats, and that occupational exposure to petroleum may result in enhanced microsomal drug metabolism.

Comparative enzyme-inducing effects of chlorpromazine and fluphenazine therapies in psychotic patients.

Antipyrine elimination kinetics were measured in psychotic patients receiving either long-term chlorpromazine or fluphenazine decanoate therapy and in non-medicated control subjects. Patients receiving chlorpromazine metabolised antipyrine faster than the controls while, in patients receiving fluphenazine decanoate, there was not change. The results suggest that long-term chlorpromazine therapy induced the activity of drug-metabolising enzymes, whereas fluphenazine decanoate therapy had no effect.

Choleretic and cholestatic effects of infused bile salts in the rat.

In rats, at low infusion rates taurocholate (TC), taurochenodeoxycholate (TCDC) and taurodeoxycholate (TCD) each produced an increase in bile flow of 20-50%. However, at high infusion rates (5-20 mumoles min-1kg-1) the cholestatic effects of the bile salts were revealed and the relative toxicity of the bile salts was seen to be TDC greater than TCDC greater than TC.

Impairment of hepatic drug metabolism in alcoholics.

1 The effects of chronic ethanol intake on the elimination kinetics of antipyrine were determined in nineteen male alcoholic subjects with comparison made to fourteen male volunteers. 2 Half-lives were longer and clearance values less in the alcoholic group. 3 Significant rank correlations were found between half-life and clearance when compared with various biochemical parameters of liver function measured in the plasma of the alcoholics. 4 These results show that a significant proportion of the alcoholics studied had impaired hepatic drug metabolizing capacity and that the activity of hepatic microsomal enzymes may be related to the extent of ethanol induced liver damage in these subjects.

Altered drug metabolism in anaesthetists exposed to volatile anaesthetic agents.

Antipyrine kinetics were measured on saliva in eight anaesthetists during a period when they were giving general anaesthetics and a period when they were working exclusively in intensive care. During the anaesthesia period there was a reduction in antipyrine half-life and the clearance of antipyrine increased. Analysis of the data in groups failed to detect these changes because of the wide variation in metabolism between subjects. Exposure to anaesthetic agents under non-scavenging operating theatre conditions appears to enhance hepatic metabolism.