Charlene A. McQueen

Suppression of Kupffer cell function prevents cadmium induced hepatocellular necrosis in the male Sprague-Dawley rat.

Exposure of humans to toxic metals and metalloids is a major environmental problem. Many metals, such as cadmium, can be hepatotoxic. However, the mechanisms by which metals cause acute hepatic injury are in many cases unknown. Previous reports suggest a major role for inflammation in acute cadmium induced hepatotoxicity. In initial experiments we found that a non-hepatotoxic dose of cadmium chloride (CdCl2; 2.0 mg/kg, i.v.) markedly increased the clearance rate of colloidal carbon from the blood, which is indicative of enhanced phagocytic activity by Kupffer cells (resident hepatic macrophages). Thus. the objective these studies was to determine the involvement of Kupffer cells in cadmium induced liver injury by inhibiting their function with gadolinium chloride (GdCl3). Male Sprague-Dawley rats were administered GdCl3 (10 mg/kg, i.v.) followed 24 h later by a single dose of CdCl2 (3.0 and 4.0 mg/kg, i.v.). Twenty four hours after CdCl2 administration animals were killed and the degree of liver toxicity was assessed using plasma alanine aminotransferase (ALT), as well as light microscopy. Cadmium chloride administration produced multifocal hepatocellular necrosis and increased plasma ALT activity. Pretreatment with GdCl3 significantly reduced both the morphological changes and hepatic ALT release caused by CdCl2. However, the protection was specific to the liver, and did not alter CdCl2 induced testicular injury, as determined by histopathological damage. In many cases, the inducible cadmium-binding protein, metallothionein (MT) is often an essential aspect of the acquisition of cadmium tolerance in the liver. Although cadmium caused a dramatic induction of hepatic MT (32-fold), GdCl3 caused only a minor increase (2-fold). Combined CdCl2 and GdCl3 treatment did not induce levels to an extent greater than CdCl2 alone. As expected, GdCl3 also caused a slight increase in the amount of cadmium associated with the liver. In cultured hepatocytes isolated from GdCl3 pretreated rats, CdCl2 induced cytotoxicity was not significantly altered compared to control hepatocytes, indicating that the mechanism of tolerance required the presence of other cell types. Thus, GdCl3 attenuation of CdCl2 induced hepatotoxicity does not appear to be caused by increased tissue MT content or a decreased susceptibility of hepatocytes to cadmium. From these data, we concluded that tolerance to cadmium induced hepatotoxicity involves the inhibition of Kupffer cell function which results in a decreased inflammatory response and an altered progression of hepatic injury. These data further indicate that Kupffer cell function is critical to cadmium induced hepatocellular necrosis.

Cytotoxicity and genotoxicity of methyleugenol and related congeners-- a mechanism of activation for methyleugenol.

Methyleugenol is a substituted alkenylbenzene found in a variety of foods, products, and essential oils. In a 2-year bioassay conducted by the National Toxicology Program, methyleugenol caused neoplastic lesions in the livers of Fischer 344 rats and B6C3F(1) mice. We were interested in the cytotoxicity and genotoxicity caused by methyleugenol and other alkenylbenzene compounds: safrole (a known hepatocarcinogen), eugenol, and isoeugenol. The endpoints were evaluated in cultured primary hepatocytes isolated from male Fischer 344 rats and female B6C3F(1) mice. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release, while genotoxicity was determined by using the unscheduled DNA synthesis (UDS) assay. Rat and mouse hepatocytes showed similar patterns of toxicity for each chemical tested. Methyleugenol and safrole were relatively non-cytotoxic, but caused UDS at concentrations between 10 and 500 microM. In contrast, isoeugenol and eugenol produced cytotoxicity in hepatocytes with LC50s of approximately 200-300 microM, but did not cause UDS. Concurrent incubation of 2000 microM cyclohexane oxide (CHO), an epoxide hydrolase competitor, with a non-cytotoxic concentration of methyleugenol (10 microM) resulted in increased cytotoxicity but had no effect on genotoxicity. However, incubation of 15 microM pentacholorophenol, a sulfotransferase inhibitor, with 10 uM methyleugenol resulted in increased cytotoxicity but had a significant reduction of genotoxicity. These results suggest that methyleugenol is similar to safrole in its ability to cause cytotoxicity and genotoxicity in rodents. It appears that the bioactivation of methyleugenol to a DNA reactive electrophile is mediated by a sulfotransferase in rodents, but epoxide formation is not responsible for the observed genotoxicity.

Prenatal expression of N-acetyltransferases in C57Bl/6 mice.

Exposure to carcinogens such as 4-aminobiphenyl (4ABP), found in tobacco smoke and other combustion products, results in the formation of detectable levels of 4ABP-hemoglobin adducts in cord blood and 4ABP-DNA adducts in conceptal tissue. The presence of these adducts requires that the parent compound undergo biotransformation. When exposure occurs in utero, the maternal, placental and conceptal tissues are all possible sites for the formation of DNA-reactive products. One step in the activation of 4ABP is catalyzed by N-acetyltransferases (NAT). The expression of NAT was evaluated in gestational day (GD) 10-18 conceptal tissues from C57Bl/6 mice. There was a quantitative increase in NAT1 and NAT2 mRNAs with increasing gestational age that was also reflected in age-related changes in functional protein measured as 4ABP-NAT activity. The ability to acetylate 4ABP increased from GD10 to 18 and was lower in conceptal tissue than in adult liver. The potential toxicologic significance of prenatal NAT expression was assessed by formation of 4ABP-DNA adducts. At GD 15 and 18, 4ABP-DNA adducts were detected by immunohistochemistry 24 h following a single oral dose of 120 mg 4ABP/kg. Based on nuclear fluorescence, conceptual 4ABP-DNA adducts were present at similar levels at GD15 and 18. Levels of 4ABP-DNA adducts were significantly higher in maternal liver compared with the conceptus. Results from this study show that both NAT genes were expressed prenatally and that functional enzymes were present. These data support the possible in situ generation of reactive products by the conceptus. The relative contributions of maternal activation of 4ABP and that by the conceptus remain to be determined.

Study of potential in vitro and in vivo genotoxicity in hepatocytes of quinolone antibiotics.

The genotoxicity of quinolone antibiotics has been evaluated in hepatocytes following in vitro and in vivo exposure. Unscheduled DNA synthesis (UDS) was induced in vitro in rat hepatocytes by norfloxacin, ofloxacin, pefloxacin, and ciprofloxacin but not by nalidixic acid. In vivo UDS was not observed in hepatocytes isolated 4 to 24 hr after exposure of adult male F344 rats to either a single dose (30 to 190 mg/kg) or repeated doses (40 mg/kg) of ciprofloxacin. Using the 32P-postlabeling technique, no modified bases were detected in hepatocytes exposed in vitro to ciprofloxacin. In summary, UDS was induced in hepatocytes by in vitro exposure to high concentrations of norfloxacin, ofloxacin, pefloxacin, or ciprofloxacin. There was no evidence of in vitro DNA adduct formation by ciprofloxacin or in vivo DNA damage under the conditions tested. These findings suggest that ciprofloxacin is not DNA reactive, but it induces in vitro UDS as a consequence of some indirect action.

The effects of genetic variation in N-acetyltransferases on 4-aminobiphenyl genotoxicity in mouse liver.

Inbred, congenic and transgenic strains of mice were characterized for acetylation of p-aminobenzoic (PABA) and the carcinogen 4-aminobiphenyl (4ABP). C57Bl/6 mice have the NAT2*8 allele, A/J mice have NAT2*9 and congenic B6.A mice have NAT2*9 on the C57Bl/6 background. The first transgenic strain with human NAT1, the functional equivalent of murine NAT2, was also tested. The murine NAT2*9 allele correlated with a slow phenotype measured with the murine NAT2 selective substrate PABA. The two strains having this allele also had a lower capacity to acetylate 4ABP. A line with five copies of the human NAT1 transgene was bred for at least five generations with either C57Bl/6 or A/J mice. There was no significant change in PABA NAT activity on the C57Bl/6 background but a 2.5-fold increase was seen in hNAT1:A/J compared with A/J. The effect of variation in NATs on 4ABP genotoxicity was assessed in these strains. Twenty-four hours after exposure to a single oral dose of 120 mg 4ABP/kg, hepatic 4ABP-DNA adducts were detected by immunofluoresence in all strains. Nuclear fluorescence intensities (mean+/-S.D.) were 41.1+/-3.6 for C57Bl/6, 37.9+/-1.11 for A/J and 36.3+/-2.44 for B6.A. There was no correlation between murine NAT2 alleles and 4ABP-DNA adduct levels. Similar results were seen with the transgenic strains. The data indicate that the range of variation present in these strains of mice was insufficient to alter susceptibility to 4ABP genotoxicity. The impact of these relatively modest differences in the acetylation of the activation of 4ABP may be masked by other competing biotransformation reactions since 4ABP is a substrate for both NAT1 and NAT2. Mouse models with variation in both isoforms are needed to adequately assess the role of variation in NATs in susceptibility to 4ABP genotoxicity.

Toxicity of the heterocyclic amine batracylin: investigation of rodent N-acetyltransferase activity and potential contribution of cytochrome P450 3A.

The heterocyclic amine, batracylin (BAT), is genotoxic and several lines of evidence suggest that acetylation is one step in the formation of a DNA-damaging product. The variation in susceptibility to BAT toxicity observed between rats and mice has also been linked to the acetylated product. BAT N-acetyltransferase (NAT) activity was determined in rat and mouse hepatic cytosols. Formation of acetylbatracylin (ABAT) was 6 times greater in F-344 hepatic samples compared to either mouse strain, while hepatic BAT NAT activities were similar in C57B1/6 and A/J mice. No deacetylation of ABAT was detected. In contrast, 2-aminofluorene NAT activity in C57B1/6 hepatic cytosol was twice that of the A/J strain and activities in both strains of mice were greater than in rat. Deacetylation of 2-acetylaminofluorene was detected in both species with enzyme activities in C57B1/6>A/J>F-344. Hepatocytes from the F-344 rats, the species most sensitive to BAT toxicity, were used to investigate the contribution of other biotransformation reactions to BAT cytotoxicity. Leakage of cellular lactate dehydrogenase was greater in hepatocytes from male rats than from females, increased on in vivo exposure to dexamethasone, and decreased in the presence of troleandomycin, suggesting that CYP3A-mediated biotransformation of BAT is involved in the formation of a cytotoxic product. When phenol red, a substrate for UDP-glucuronsyltransferase (UDPGT), was absent from the medium, BAT cytotoxicity was reduced. These data are consistent with a role for NAT, CYP, and UDPGT in the biotransformation of BAT.

An N-ethyl-N-nitrosourea-induced mutation in N-acetyltransferase 1 in mice

Genetic variation in human N-acetyltransferases (NAT) has been implicated in susceptibility to aromatic amine and hydrazine carcinogens and therapeutic drugs. There are mouse models for variability of human NAT1; however mice with genetic differences in Nat1 (corresponding to human NAT2), have not been available. N-Ethyl-N-nitrosourea (ENU) mutagenesis was used to create genetic variation in Nat1. Among a number of mutations identified, a base-pair change substituting threonine for isoleucine at position 95 was recovered and studied. Molecular models suggested that this substitution would alter substrate binding. Analysis of hepatic Nat1 activity with the selective substrate isoniazid showed that there was a significant reduction in enzymatic activity in the homozygous mutants compared to the parental strain.

Measuring the Activity of Arylamine N‐Acetyltransferase (NAT)

This unit describes methods for measuring the activity of arylamine N‐acetyltransferases (NAT). Genetic polymorphisms in NAT 1 and NAT 2 have been associated with susceptibility to aromatic amines carcinogens and effects of therapeutic drugs. Evaluation of the activities associated with substrates of NATs is helpful in elucidating the contribution of these enzymes to the pharmacologic and toxicologic effects of arylamines and hydrazines.