Hepatic Macromolecules Research Articles

Thioacetamide – inhalable fraction. Documentation of proposed values of occupational exposure limits (OELs)

Thioacetamide occurs in the form of colorless crystals with a characteristic smell of mercaptans. It was used in the past as a fumigant to prevent oranges from rotting, in rubber vulcanization and as a diesel stabilizer. It is currently used in a qualitative analysis as a source of hydrogen sulfide. According to information from the Central Register of Data on Exposure to Carcinogenic or Mutagenic Substances, Mixtures, Factors or Technological Processes in 2005-2016 from 486 to 1137 people were exposed to thioacetamide in Poland. Most of them were women. The LD50 value after intragastric administration of the compound to rats is 301 mg/kg. Thioacetamide is a strong hepatotoxic agent, its single dose caused hepatic necrosis. Administered repeatedly it induced liver damage, which was indicated by biochemical changes and cirrhosis. The effects of thioacetamide toxicity in chronic animal experiments indicated a relationship to exposure time. After chronic exposure of rats to thioacetamide in drinking water (at 0.03%, i.e., approximately 35 mg/kg/day) or in feed (0.5% in feed, i.e., approximately 28 mg/kg/day), hepatitis and local hepatic foci were noted after 4 months, these changes later intensified, and after 8–17 months chronic hepatitis, cirrhosis and cancer of the liver and bile ducts occurred. The results of mutagenicity and genotoxicity studies of thioacetamide are inconclusive. It can be assumed that the compound may damage genetic material in vivo after biotransformation to a highly hepatotoxic metabolite. The metabolism of thioacetamide by S-oxidation (mainly with the participation of CYP2E1) leads to the production of sulfoxide (TASO), and then to hepatotoxic, highly reactive sulfone (TASO2). The latter is of fundamental importance for the mechanism of toxic action of thioacetamide (by binding with hepatic macromolecules). Thioacetamide metabolites also induce oxidative stress. Because of neoplasms observed in chronic studies, International Agency for Research on Cancer (IARC) included thioacetamide in group 2B – agents probably carcinogenic to humans. According to the CLP classification, thioacetamide is a category-1B carcinogen with a "H350 – May cause cancer" note. The hepatotoxic effects of thioacetamide in rats after repeated administration were used as the basis for determining the maximum acceptable concentration (MAC; TLV-TWA – threshold limit value-time weighted average). A concentration of 1.5 mg/m3 was proposed as the MAC value. There are no bases to determine the short-term exposure limit (STEL) and the biological limit value (BLV). "Carc. 1B" marking is also proposed, as thioacetamide is a category-1B carcinogen. This article discusses the problems of occupational safety and health, which are covered by health sciences and environmental engineering.

EVALUATION OF THE HEALTH RISK ASSOCIATED WITH EXPOSURE TO CHLOROFORM IN INDOOR SWIMMING POOLS

The exposure of swimmers to chloroform (CHCl5) was investigated in indoor swimming pools of the Quebec City region along with the associated carcinogenic risk. Six training sessions involving 52 competition swimmers (11 to 20 yr old) were conducted in 3 different pools, while 12 adult leisure swimmers attended 5 sessions, each held in a different pool. For each session, water and ambient air CHCl3 concentrations were measured and CHCl3 levels in alveolar air samples (CHCl3ALV) collected from swimmers prior to entering the swimming pool premises and after 15, 35, and 60 min of swimming. Mean water concentrations varied from 18 µg/L to 80 µg/L, while those in air ranged from 78 µg/m3 to 329 µg/m3. Multiple linear regression analyses revealed that CHCl3ALV values in competition swimmers were strongly correlated to ambient air and water levels, and to a lesser degree to the intensity of training. Only ambient air concentration was positively correlated to CHCl3ALV in the leisure group. Concentrations of CHCl3 metabo lites bound to hepatic and renal macromolecules, estimated using a physiologically based pharmacokinetic (PBPK) model, were 1.6 and 1.9 times higher for the competition swimmers than for the leisure swimmers, respectively. The highest hepatic concentration predicted in competition swimmers, 0.22 µg CHCl equivalents/kg of tissue, was at least 10,000 times lower than the smallest no observed3effect level for liver tumors in animals. Data indicate that the safety margin is therefore very large, for competitive swimmers as well as for leisure swimmers.

Genetic predisposition to drug-induced hepatotoxicity

Drug-induced hepatitis is uncommon and generally unpredictable. Hepatotoxicity may be related to the drug itself, or to chemically reactive metabolites which can bind covalently to hepatic macromolecules and may lead to either idiosyncratic, toxic hepatitis or to immunoallergic hepatitis. There is now evidence indicating that genetic variations in systems of biotransformation or detoxication may modulate either the toxic or sensitizing effects of some drugs. Thus, the genetic deficiency in a particular hepatic cytochrome P 450 isozyme (CYP 2D6) is involved in perhexiline liver injury. The deficiency in CYP 2C19 might also contribute to Atrium hepatotoxicity. Slow acetylation related to N-acetyltransferase 2 deficiency contributes to sulfonamide hepatitis. The genetic deficiency in glutathione synthetase may increase the susceptibility to several drugs including acetaminophen. A constitutional deficiency in another cell defense mechanism, still not characterized, seems to increase significantly the risk of hepatotoxicity with halothane, phenytoin, carbamazepine, phenobarbital, sulfamides and amineptine.

Determination of Aflatoxin B1Biotransformation and Binding to Hepatic Macromolecules in Human Precision Liver Slices

Although epidemiological studies suggest that aflatoxin B1(AFB1) is a human carcinogen, at least in the presence of hepatitis B virus infection, animal studies have demonstrated large differences in species sensitivity to AFB1, and the sensitivity of humans relative to experimental animals remains unclear. The purpose of this study was to determine the profile of AFB1metabolism and the extent of AFB1binding to cell macromolecules in human liver slices under experimental conditions that would allow direct comparison to similar endpoints in the rat, a species sensitive to the carcinogenic actions of AFB1. Liver slices were prepared from three individual human liver samples with a Krumdieck tissue slicer and incubated with 0.5 μM[3H]AFB1for 2 hr. Significant interindividual variations were observed in the rates of oxidative metabolite formation and in specific binding to cell macromolecules. The rates of oxidative metabolism of AFB1to AFQ1, AFP1, and AFM1in the three human liver samples were similar to those previously observed in rat liver slices. AFB1–GSH conjugate formation was not detected in any of the human liver samples, and yet specific binding of AFB1to cell macromolecules was considerably lower in the human liver slices relative to that in rat liver slices. AFB1–DNA binding levels ranged from 3 to 26% of control rat and AFB1–RNA binding levels ranged from 25 to 49% of control rat. The AFB1–protein binding level in the one human sample measured was 20% of that observed for control rat. While these results suggest that humans do not form as much AFBO as the rat, they are also consistent with the hypothesis that humans do not possess GST isozyme(s) with high specific activity toward AFBO. Significant individual differences in AFB1metabolism and binding between humans suggest the presence of genetic and/or environmental factors that may confer large variability in susceptibility to AFB1.

Mutagenicity and metabolism of dimethyl phthalate and its binding to epidermal and hepatic macromolecules

As an active ingredient in insect repellents, dimethyl phthalate (DMP) had previously been shown to produce chromosomal aberrations in the livers of rats following subchronic application of the phthalate to skin. When we tested DMP in the Ames mutagenesis assay, it produced in bacterial tester strain TA100 (but not TA98) a dose-related mutagenic response that was abolished by NAD- and NADP-independent metabolism associated with rat liver microsomal preparations (S9). In a host-mediated mutagenesis assay, rats were injected ip with DMP (2 g/kg body weight); urine was collected for 24 h, extracted, and analyzed for mutagenic activity and phthalic acid-containing derivatives. The extracted urine was not mutagenic to TA100 and contained an equivalent of 1.96 mg phthalate/ml urine. More than 97% of the phthalic acid-containing derivatives present in the extracted urine consisted of the nonmutagenic metabolite of DMP, monomethyl phthalate (MMP). In vitro experiments showed that rat liver homogenates hydrolyzed 93% of carbonyl-labeled 14C-DMP (7.7 mM) to MMP in 2 h and bound 0.07 nmol of [14C]phthalate/mg liver macromolecules. By contrast, rat epidermal homogenates metabolized only 5% and bound 38-fold higher levels of carbonyl-labeled 14C-DMP (2.66 nmol/mg of macromolecules), with no detectable binding to nucleic acids. Compared to epidermis and plasma, liver had a fivefold higher rate of DMP monoesterase activity (1240 nmol/h/mg protein), which, when inhibited by 67%, resulted in a 4.4-fold increase in phthalate-bound hepatic macromolecules (0.31 vs. 0.07 nmol of carbonyl-labeled 14C-DMP/mg macromolecules). In addition to MMP, formaldehyde was produced during the metabolism of DMP by liver. When ethanol was used to inhibit the oxidation of DMP-derived methanol by hepatic homogenates, there resulted a 74% reduction in the accumulation of formaldehyde and similar reductions of 71 and 73% in the binding of methyl-labeled 14C-DMP to nucleic acids and macromolecules. (Methyl-labeled, unlike carbonyl-labeled, 14C-DMP yields a 14C-labeled methanol when hydrolyzed.) These results indicate that the DMP diester is a weak bacterial mutagen, which binds to epidermal and hepatic macromolecules other than nucleic acids, and that although the rapid hepatic metabolism of DMP to its monoester (MMP) and methanol affords protection against higher levels of phthalate binding as well as against DMP-induced bacterial mutagenesis, it also oxidizes DMP-derived methanol to formaldehyde, a metabolite that binds macromolecules, including nucleic acids.

Hepatic macromolecular binding and tissue distribution of ortho- and para-toluidine in rats

The in vivo covalent binding of ortho- and para-toluidine (OT and PT) to rat hepatic macromolecules was investigated to determine if a relationship exists between the degree of binding for each isomer and its carcinogenic potency. The ortho-isomer has been shown to be a more potent hepatocarcinogen than the para-isomer. In addition to the macromolecular binding, the tissue distribution of each isomer was also measured. The degree of binding to hepatic macromolecules appeared to be at maximum for both at 24–28 h following dosing. At 24 h following dosing, the level of DNA binding of OT was approximately 1.2-fold lower than that of PT. The binding to RNA and protein was also lower for OT than PT, although the differences were not as great as that observed for DNA binding. There were subtle differences in tissue distribution for each isomer. However, in contrast to the macromolecular binding data, the area under the plasma concentration curve for OT was approximately 1.8-fold greater than that for PT. Based on the results of these studies, there was no direct correlation between the degree of macromolecular binding and carcinogenic potency.

Covalent interactions of 1,2,3-trichloropropane with hepatic macromolecules: Studies in the male F-344 rat

Preliminary investigations into the role of biotransformation in 1,2,3-trichloropropane (TCP)-induced tumor formation have been undertaken. Male F-344 rats were administered 30 mg/kg [ 14C]TCP (100 μCi/kg) ip and killed 4 hr later. The extent of covalent binding to hepatic protein, DNA, and RNA was 418, 244, and 432 pmol [ 14C]TCP equivalents/mg, respectively. An in vivo covalent binding time course showed no significant change in [ 14C]TCP equivalents bound to hepatic DNA (1–48 hr), while binding to protein was maximal by 4 hr and decreased significantly by 48 hr. The binding of TCP-associated radioactivity to hepatic protein and DNA was shown to be cumulative for two and three doses when given 24 hr apart. Pretreatment of animals with phenobarbital caused a decrease while pretreatment with SKF 525-A caused an increase in covalent binding of [ 14C]TCP equivalents to protein and DNA. Pretreatment of rats with β-naphthoflavone did not alter the covalent binding of [ 14C]TCP equivalents to protein or DNA. However, glutathione depletion with l-buthionine-( R,S)-sulfoximine increased binding to protein by 342% while it decreased binding to DNA by 56%. Intraperitoneal administration of TCP also depleted hepatic GSH by 41 and 61% 2 hr after doses of 30 and 100 mg/kg. The in vivo binding data suggest a dual role for GSH in the bioactivation of TCP. It may, in part, be that GSH is involved in the bioactivation and covalent binding of TCP to hepatic DNA. However, it also appears to detoxify a reactive intermediate(s) that binds to protein.

Modulation of cocaine metabolism in primary rat hepatocyte cultures: Effects on irreversible binding and protein biosynthesis

To study mechanisms of cocaine-induced hepatotoxicity, short-term-cultured rat hepatocytes were exposed to cocaine or norcocaine at 10 −6 to 10 −4 m. Induction in vivo (with Aroclor 1254) or inhibition in vitro (with SKF-525A) of cytochrome P450 modulated the rate of oxidative biotransformation of cocaine to norcocaine and to other metabolites in vitro. The quantitative changes in the metabolic conversion of cocaine were paralleled by the amount of radiolabeled cocaine equivalents irreversibly bound to hepatocellular protein. Furthermore, induction of cytochrome P450-mediated cocaine or norcocaine metabolism was associated with inhibition of protein biosynthesis in cultured hepatocytes, whereas this effect was restored to normal when the oxidative metabolism was blocked by SKF-525A. Glutathione depletion with buthionine sulfoximine both increased the covalent binding of cocaine to hepatic macromolecules and augmented the inhibitory effect on protein biosynthesis. The integrity of the hepatocellular plasma membrane was not affected (no effect on lactate dehydrogenase leakage). The results indicate that in rat hepatocytes (a) a high proportion of intracellular cocaine is converted to a reactive metabolite which irreversibly binds to protein, and (b) irreversible binding of cocaine to hepatic protein is associated with impairment of hepatocellular function and could play a role in cocaine-mediated hepatotoxicity.

Formation of ortho-phenylphenol glutathione conjugates in the rat liver.

1. The radioactive materials(s) derived from 14C-ortho-phenylphenol (OPP) became bound irreversibly to hepatic microsomal macromolecules in an NADPH-generating system, and the binding was inhibited by cysteine and glutathione (GSH). 2. A water-soluble phenyl-hydroquinol (PHQ)-GSH conjugate was produced by non-enzymic reaction between phenyl-p-benzoquinone and GSH in alcoholic aqueous solution, and the structure of the conjugate was confirmed by its 13C-n.m.r. spectrum. 3. When 14C-OPP and GSH were incubated in a microsomal NADPH-generating system, radioactive material derived from the aqueous phase of the incubation mixture was similar to the synthetic PHQ-GSH on t.l.c. 4. PHQ-GSH was excreted as a minor conjugate into the bile after oral administration of OPP (1000 mg/kg) to rats, the cumulative biliary excretion of the conjugate was about 4% of the dose in 6 h.

Effects of single-dose and repeated-dose pretreatment with 2(3)-tert-butyl-4-hydroxyanisole (BHA) on the hepatobiliary disposition and covalent binding to DNA of aflatoxin B 1 in the rat

The effects of two distinctive BHA pretreatment regimens on the biliary excretion of aflatoxin B 1 (AFB) metabolites and the covalent binding of AFB to hepatic DNA were studied in vivo in the rat. To differentiate between enzyme induction effects and direct antioxidant effects, BHA was given to rats for 9 days (500 mg/kg/day, sc) or as a single dose (500 mg/kg, po), and [ 3H]AFB was administered ip. Repeated treatment with BHA enhanced the biliary excretion of both the glutathione conjugate of AFB and the AFP 1-glucuronide to 200% of control values, reduced the amount of AFB remaining in the liver to 53% of control and reduced the covalent binding of AFB to hepatic DNA to 16% of control. A single BHA treatment had no effect on the biliary excretion of AFB or the binding of AFB to hepatic macromolecules, even though high concentrations of BHA were present in the liver during the period of AFB metabolism. These results support the hypothesis that BHA inhibits AFB carcinogenesis via the induction of phase II biotransformation pathways such as glutathione S-transferase, which act to reduce the amount of AFB-epoxide available for binding to DNA. We found no evidence of a direct antioxidant effect of BHA in altering the hepatobiliary disposition of AFB.

The effect of diet on 2,6-dinitrotoluene hepatocarcinogenesis.

Pectin-induced changes in microflora have been shown to elevate the covalent binding of 2,6-dinitrotoluene (2,6-DNT)-related materials to total rat hepatic macromolecules. Therefore, the effect of diets varying in pectin content on the induction of foci and hepatic tumors induced by 2,6-DNT was studied in male F344 rats. 2,6-DNT (3.0-3.5 and 0.6-0.7 mg/kg/day) was incorporated into NIH-07 (NIH), an open formula cereal-based diet high in pectin content, AIN-76A (AIN), a purified pectin-free diet, or AIN-76A supplemented with 5% pectin (AP). Hepatic foci were scored after histochemical staining for gamma-glutamyl transpeptidase (GGT), canalicular adenosine triphosphatase or glucose-6-phosphatase following administration of test diets for 3, 6 and 12 months. The number of foci per cm3 of liver increased in a dose- and time-department manner following incorporation of 2,6-DNT into test diets with NIH greater than AP greater than AIN. In the NIH diet, 2,6-DNT did not alter the phenotypic distribution of foci. Animals fed control or 2,6-DNT-containing AIN and AP diets had few or no GGT foci throughout the study. Hepatocellular carcinomas and neoplastic nodules were observed only in rats fed NIH containing 2,6-DNT. The concentrations of 2,6-DNT-related material covalently bound to hepatic macromolecules after a single oral dose of radiolabeled 2,6-DNT given after 12 months on the diets increased in control rats and in rats receiving low dose 2,6-DNT in the diet with AIN less than AP less than NIH. These studies show that the carcinogenicity of 2,6-DNT differs depending on whether rats are fed an NIH or AIN (+/- pectin) diet. The results suggest that diet-induced alterations in the covalent binding of 2,6-DNT are not the sole factor in determining the carcinogenic response to 2,6-DNT. Furthermore, unidentified contaminants in cereal-based diets may influence foci and tumor production in rat liver during carcinogen treatment.

Species differences in the toxicity of p-chloro- o-toluidine to rats and mice. Covalent binding to hepatic macromolecules and hepatic non-parenchymal cell DNA and an investigation of effects upon the incorporation of [ 3H] thymidine into capillary endothelial cells

The interaction of p-[ 14C] chloro- o-toluidine with hepatic macromolecules of rats and mice has been investigated. At all time points after single administration the extent of binding decreased in the order protein > RNA > DNA in both species. The level of binding to mouse liver DNA was greater than that to rat liver DNA after both single and repeated administration. In vitro studies showed that mouse liver fractions catalysed the binding of p-chloro- o-toluidine to calf thymus DNA more readily than rat liver fractions. Conversely, binding to protein and RNA was more marked in the rat than in the mouse. Species differences in DNA repair rates were not observed. The results failed to demonstrate a preferential persistence of binding to mouse liver non-parenchymal cell DNA. Autoradiographic determinations did not demonstrate any effect of p-chloro- o-toluidine upon the incorporation of [ 3H] thymidine into subcutaneous capillary endothelial cells. The results suggest that different reactive metabolites are responsible for binding to DNA and protein, and that the pattern of reactive metabolites formed from p-chloro- o-toluidine in the mouse differs from that formed in rats.

Formation of DNA adducts in vivo in rat liver and intestinal epithelium after administration of the carcinogen 3,2'-dimethyl-4-aminobiphenyl and its hydroxamic acid.

Administration of the 3H-labeled colon carcinogen, 3,2'-dimethyl-4-aminobiphenyl (DMABP) and its hydroxamic acid derivative, N-hydroxy-N-acetyl-DMABP, to male F344 rats resulted in high levels of covalent binding to hepatic and intestinal DNA, RNA and protein. For both compounds, binding to hepatic macromolecules was 2-4 times higher than in the intestine. High pressure liquid chromatographic analysis of the enzymatically hydrolyzed DNA from liver and intestinal epithelium indicated the presence of two carcinogen-DNA adducts: 5-(deoxyguanosin-N2-yl)-DMABP (15%), N-(deoxyguanosin-8-yl)-DMABP (50%), and a decomposition product of the latter (15%). N-acetylated adducts were not detected. When measured after 7 days, all adducts in the intestinal DNA had decreased by 70%, while only a 29% decrease had occurred in the hepatic DNA. To determine if the loss of DMABP products was a consequence of cell turnover or repair, rats were treated with [3H]thymidine and DMABP, and the specific activity of hepatic liver and intestinal DNA was measured. Between 1 and 7 days only a slight decrease in [3H]thymidine content occurred in hepatic DNA as compared with a 95% reduction in intestinal DNA. Thus, the higher rate of DNA synthesis in the intestine versus that in the liver may serve to promote fixation of the initiating lesion and account for the preferential induction of intestinal cancer by DMABP. Furthermore, comparison of these data with metabolic activation pathways reported earlier strongly suggest that N-hydroxy-DMABP is the proximate carcinogenic metabolite of both DMABP and N-hydroxy-N-acetyl-DMABP.

Hepatic macromolecular covalent binding of mononitrotoluenes in Fischer-344 rats

The mononitrotoluenes are important industrial chemicals which display isomeric specificity in their ability to induce hepatic DNA excision repair in Fischer-344 rats. Covalent binding of the structurally related hepatocarcinogen, 2,6-dinitrotoluene, to hepatic DNA is markedly decreased by prior administration of the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The objectives of this study were to determine whether hepatic macromolecular covalent binding of the mononitrotoluene isomers differed and to determine whether covalent binding of the mononitrotoluenes to hepatic DNA in vivo was decreased by inhibitors of sulfotransferase. Male Fischer-344 rats were given a single oral dose of [ ring-U- 14C]-2-, 3- or 4-nitrotoluene (2-, 3- or 4-NT) and killed at various times thereafter. Livers were removed and analyzed for total and covalently bound radiolabel. Maximal concentrations of total radiolabel were observed between 3 and 12 h after the dose, and there were no large differences among the 3 isomers in peak concentrations achieved. Covalent binding to hepatic macromolecules was maximal 12 h after administration for all three isomers. Thereafter, concentrations of covalently bound 2-NT-derived material were always 2–6 times higher than those of 3- or 4-NT-derived material. When DNA was isolated from livers of rats given the mononitrotoluenes 12 h previously, only 2-NT was observed to covalently bind at concentrations above the limits of detection of the assay. The covalent binding of 2-NT, but not that of 3- or 4-NT, to both total hepatic macromolecules and DNA was markedly decreased by prior administration of either PCP or DCNP. Covalent binding to hepatic DNA was decreased by >96%. The results of this study correlate well with studies which have demonstrated that 2-NT, but not 3- or 4-NT, induces DNA excision repair. Furthermore, they suggest that 2-NT, like the hepatocarcinogen 2,6-dinitrotoluene, requires the action of sulfotransferase for its conversion to a species capable of covalently binding to hepatic DNA.

Metabolism of 1-[ 14C]nitropyrene in isolated perfused rat livers

1-Nitropyrene (1-NP), a constituent of diesel exhaust, is carcinogenic to rats and is a bacterial and mammalian mutagen. Biliary and fecal excretion of 1-NP metabolites are the major routes of excretion in rats, suggesting that hepatic metabolism plays a dominant role in determining the biological fate of 1-NP. The purpose of this investigation was to quantitate 1-[ 14C]NP metabolites formed in isolated perfused rat livers and excreted in bile from rats. Perfused rat livers displayed a capacity for oxidation, reduction, acetylation, and conjugation of 1-NP (or its metabolites). Reduction of 1-NP followed by N-acetylation was the major metabolic pathway observed in the perfused livers. Acetylaminopyrene (AAP) was the major metabolite detected, with total quantities (150 nmol) accounting for about 60% of the total 1-[ 14C]NP dose (258 nmol) added to the perfusate. Considerably smaller quantities of aminopyrene and hydroxynitropyrenes were also detected. Livers perfused with 1-[ 14C]NP excreted about 36 nmol equivalents of 1-[ 14C]NP (12% of the total 1-NP dose) in bile after 60 min. Some of the biliary metabolites were tentatively identified as metabolites of the mercapturic acid pathway. The spectrum of biliary metabolites was qualitatively identical to that seen in bile from intact rats. Quantities of 14C covalently bound to hepatic macromolecules from perfused livers were 0.4 nmol 1-NP eq/g liver. The data from this study indicate that the liver may be an important site for metabolism of 1-NP.

Autoradiographic and biochemical studies of drug distribution in the liver. III. [14C]Aminotriazole.

Whole body autoradiography revealed that the distribution pattern of [14C]aminotriazole in the mouse liver was homogeneous after intravenous administration of the labeled agent and then became heterogeneous (or reticular). Microautoradiography by dry-mounting method revealed that the macroscopic heterogeneous pattern was due to the central localization of the radioactive compound in the hepatic lobule. The present studies indicated that the heterogeneous distribution could be explained as follows. The amount of [14C]aminotriazole circulated to the liver was large since the compound was not so significantly distributed in non-hepatic tissues: distribution pattern was homogeneous in the liver. This was shown by whole body autoradiography and radiometry of tissues. A part of [14C]aminotriazole radioactivity present in the liver was gradually bound covalently to hepatic macromolecules. This was shown by whole body autoradiography after whole body sections of the mouse were extracted by acid, and by the biochemical fractionation of the liver. The covalently bound radioactivity alone became apparent in centrilobular hepatocytes: the distribution was heterogeneous. This was shown by microautoradiography and by the finding that the elimination rate of the bound radioactivity was slower than that of unbound radioactivity.

2-Hexanone potentiation of [ 14C]chloroform hepatotoxicity: Covalent interaction of a reactive intermediate with rat liver phospholipid

Rats were treated with [ 14C]chloroform ( 14CHCl 3) in corn oil (CO) or corn oil alone 18 hr following pretreatment with 2-hexanone (2-HX) in corn oil or corn oil alone. Livers were removed, homogenized 1, 2, and 6 hr post- 14CHCl 3 administration, and glutathione (GSH) content, irreversible binding of 14CHCl 3-derived radiolabel, and phospholipid composition were determined. The combination of 2-HX + CHCl 3 reduced GSH content to 21% of control (CO + CO) 1 hr after CHCl 3 administration. No significant rebound of GSH was observed 24 hr post-CHCl 3 administration. In contrast, GSH was not altered by administration of CHCl 3 to CO-pretreated rats. Although 14CHCl 3-derived radiolabel was irreversibly bound to hepatic macromolecules of both CO- and 2-HX-pretreated rats, total irreversibly bservation was consistent with the decrease in GSH of 2-HX-pretreated rats. Total 14C binding in 2-HX-pretreated rats reached a plateau 2 hr post- 14CHCl 3 administration and was distributed 52% in protein, 41% in lipid, and 7% in acid soluble fractions 6 hr post- 14CHCl 3 administration. 2-HX enhanced 14C binding to protein and lipid at each time point. Radiolabel was not detected in neutral lipids of control or 2-hexanone-treated animals, but was enhanced 33-fold in phospholipids of 2-hexanone-treated animals. Phospholipid fatty acid methyl ester derivatives did not contain 14C indicating the radiolabel was most likely associated with phospholipid polar head groups. Two dimensional thin layer chromatographic analysis of phospholipid from treated animals demonstrated that 87% of the total radiolabel was associated with a specific phospholipid ( 14C-PL) which had a 1:1 molar ratio of phosphate to 14C. The latter indicates that the 14C-PL was a monophospholipid derivative of 14CHCl 3 reactive intermediate, generally thought to be phosgene. Concurrent decrease in phosphatidylethanolamine content from 23% of total phospholipid to 7%, accumulation of 14C-PL to 2.6% of total phospholipid, and increase in lysophosphatidylethanolamine from 1 to 7% of total phospholipid durkng 2-hexanone + 14CHCl 3 treatment indicated that the amine moiety of phosphatidylethanolamine polar head groups was the probable target of phosgene-lipid interaction, and that a degradative pathway existed which removed the abnormal phospholipid from hepatic membranes. No phospholipid other than phosphatidylethanolamine was depleted. During model studies, 12% phosgene in toluene was reacted with liver phosphatidylethanolamine for 6 hr at 37°C. Two reaction products were formed, further substantiating that phosphatidylethanolamine (PE) may react with [ 14C]phosgene in liver. Although the data presented here are insufficient to conclude that the depletion of PE is a major mechanism in CHCl 3 hepatotoxicity, documented studies in which altered phospholipid composition modified cellular function suggest the possibility that PE depletion may contribute to phosgene-induced liver injury.

Indole-3-carbinol protects against covalent binding of benzo[ a]pyrene and N-nitrosodimethylamine metabolites to mouse liver macromolecules

Benzo[ a]pyrene (BaP) and N-nitrosodimethylamine (NDMA) are carcinogens and indirect acting mutagens. A naturally occurring dietary indole, indole-3-carbinol (I-3-C), has been shown to decrease the incidence of aryl hydrocarbon induced neoplasia in experimental animals. We examined the relationship between the ability of I-3-C to alter the rate of carcinogen oxidation and its ability to decrease the rate of covalent binding of carcinogen metabolites to DNA and protein. We found that I-3-C inhibited the covalent binding of NDMA oxidation products to DNA in vitro in proportion to its ability to inhibit carcinogen metabolism. Pretreatment of mice by gavage with I-3-C resulted in no change in the rate of aryl hydrocarbon hydroxylase or NDMA demethylase in hepatic post-mitochondrial supernatant. However, this pretreatment resulted in a 60–90% decrease in the ability of carcinogen oxidative metabolites to bind covalently to DNA or protein in vitro. Similarly, in in vivo experiments, gavage with I-3-C, followed by gavage with BaP or NDMA, resulted in a 63–85% decrease in covalent binding to macromolecules, with no concomitant change in carcinogen metabolism. The results suggest that the in vivo administration of I-3-C may confer protection for hepatic macromolecules against covalent binding of the metabolites of these two indirect acting mutagens.

Effects of chronic exposure to aflatoxin B1 and aflatoxin M1 on the in vivo covalent binding of aflatoxin B1 to hepatic macromolecules.

Induction of resistance to aflatoxin B1 (AFB1) binding to cellular macromolecules in the rat by chronic exposure to AFB1 and aflatoxin M1 (AFM1) was investigated. The binding of [14C]AFB1 to liver macromolecules was measured in F-344 rats fed 0.5 ppb or 50 ppb AFM1 or 50 ppb AFB1 for 41 wk. The animals then received an intragastric dose of [14C]AFB1 at 5 micrograms/kg and were sacrificed 6 h later. Hepatic DNA, RNA, and protein were isolated by chloroform-phenol extraction and hydroxylapatite chromatography. In animals preexposed to 50 ppb AFB1, labeled AFB1 binding to DNA, RNA, and protein was decreased by 72%, 74%, and 61%, respectively. Preexposure to AFM1 resulted in a small reduction in binding to nucleic acids. Glutathione transferase activity was increased by 133% in animals fed 50 ppb AFB1, by 48% in those preexposed to 50 ppb AFM1, and remained at control values in rats fed 0.5 ppb AFM1. These results suggest that the induction of detoxification enzymes following chronic exposure to aflatoxin might contribute to the reduction in covalent binding of AFB1 to macromolecules.

Circadian rhythm in acetaminophen toxicity: Role of nonprotein sulfhydryls

A circadian rhythm in the toxicity of acetaminophen administered at different times of the day was exhibited in male mice. Peak lethality (70% dead) occurred at 1800 hr and nadir lethality (10% dead) occurred at 1000 hr. Hepatic nonprotein sulfhydryl (NPS) concentrations also varied in a circadian manner and were found to exhibit an inverse relationship to the acetaminophen lethality rhythm, with the highest hepatic concentrations occurring at 1000 hr and the lowest concentrations occurring at 1800 hr. At 1800 hr when acetaminophen lethality was greatest and NPS levels the lowest, more reactive acetaminophen metabolites were found covalently bound to hepatic macromolecules as compared to those bound at 1000 hr when NPS levels were highest and acetaminophen lethality was lowest. Hepatic cytochrome P-450 levels measured at these two daily time periods did not differ. The plasma half-life of acetaminophen was significantly decreased at the 1000 hr time period as compared to the 1800 hr time period. These findings suggest that the normal daily changes in NPS levels may influence the lethality of acetaminophen.