Lack of effect of coumarin on unscheduled DNA synthesis in the in vivo rat hepatocyte DNA repair assay.

The ability of coumarin to induce UDS in male Sprague-Dawley CD rat hepatocytes in vivo was assessed using the unscheduled DNA synthesis (UDS) assay. From a preliminary toxicity study the oral maximum tolerated dose (MTD) of coumarin was determined to be 320 mg/kg body weight. For the UDS studies, rats were treated with 0 (corn oil control), 32 (one-tenth the MTD), 107 (one-third the MTD) and 320 (MTD) mg/kg coumarin via oral gavage. Rats were also treated with 20mg/kg body weight dimethylnitrosamine (DMN) or 50mg/kg body weight 2-acetylaminofluorene (2-AAF) as positive controls for the 2-4 hr and 12-16 hr expression of UDS, respectively. Hepatocytes were isolated by liver perfusion either 2-4 hr or 12-16 hr after treatment and cultured in medium containing [methyl-(3)H]thymidine for 4 hr and assessed for UDS by grain counting of autoradiographs. Coumarin treatment at doses of 32-320 mg/kg body weight had no statistically significant or dose-related effect on UDS in rat hepatocytes either 2-4 hr or 12-16 hr after dosing. In contrast, both DMN 2-4 hr after dosing and 2-AAF 12-16 hr after dosing produced significant increases in UDS assessed as the net nuclear grain count. Both genotoxins also increased the percentage of hepatocyte nuclei with greater than 5 net grains. Treatment with coumarin, DMN and 2-AAF had no statistically significant effect on the proportion of rat hepatocytes undergoing replicative DNA synthesis. In summary, this study demonstrates that coumarin does not induce UDS in hepatocytes of male Sprague-Dawley CD rats after oral administration at doses up to the MTD of 320 mg/kg. The responsiveness of the animals used in this study to genotoxic agents was demonstrated by the clear induction of DNA repair after treatment with DMN and 2-AAF.     

Assessment of unscheduled and replicative DNA synthesis in hepatocytes treated in vivo and in vitro with unleaded gasoline or 2,2,4-trimethylpentane

In a recent chronic inhalation exposure study, unleaded gasoline (UG) produced kidney tumors in male rats and liver tumors in female mice, but did not increase the incidence of liver tumors in male mice or rats of either sex. To examine the possible basis for this pattern of hepatocarcinogenesis, unscheduled DNA synthesis (UDS) as an indicator of genotoxic activity and replicative DNA synthesis (RDS) as an indicator of cell proliferation were measured in rat and mouse hepatocytes following in vivo and in vitro exposures to UG and 2,2,4-trimethylpentane (TMP), a nephrotoxic component of UG. Primary hepatocyte cultures, prepared from cells isolated from Fischer-344 rats, B6C3F1 mice, or human surgical material, were incubated with [3H]thymidine and the test agent. UDS was measured by quantitative autoradiography as net nuclear grains (NG). By similar methods, UDS and RDS (S-phase cells) were measured in hepatocytes isolated from rats and mice treated by gavage with TMP (500 mg/kg) or UG (100 to 5,000 mg/kg). A dose-related increase in UDS activity was observed in rat hepatocytes treated in vitro with 0.05 to 0.10% (v/v) UG. These doses were, however, toxic in both mouse and human hepatocyte cultures. Weak UDS activity was observed in hepatocytes isolated from male and female mice treated 12 hr previously with UG. No UDS was induced in rat hepatocytes treated in vivo or in vitro with TMP. Twenty- and fourfold increases in the percentage of cells in S-phase were observed 24 hr after treatment with TMP in male and female mice, respectively, as compared to a fivefold increase in male rats. UG increased the percentage of S-phase cells in male mice by ninefold but failed to induce RDS in females. Thus, there appears to be genotoxic compounds in UG that can be detected in cultured hepatocytes and in the livers of exposed mice. The lack of UDS activity in rat liver was consistent with the reported lack of liver tumors in chronically exposed rats. However, neither the UDS nor the RDS responses in mice exposed by gavage correlated to the sex-specific pattern of liver tumors observed in the 2-year bioassay.     

Pyridine does not induce unscheduled DNA synthesis (UDS) in hepatocytes of male B6C3F1 mice treated in vivo

Pyridine was evaluated in an in vivo/in vitro mouse DNA repair assay. Unscheduled DNA synthesis (UDS) was used as an indicator of DNA damage to hepatocytes from male B6C3F1 mice. Test animals were exposed by oral gavage to pyridine or to the vehicle or positive control articles, and hepatocytes were collected and labeled by incubation in media supplemented with [3H]thymidine. Following labeling, the cultures were processed for autoradiographic analysis. Doses were selected based on a pilot study in which 0, 250, 500, 750, 1000 or 2000 mg kg(-1) pyridine in water was administered by gavage. Mice in the 1000 and 2000 mg kg(-1) dose groups were comatose following dosing and died within 24 h of dose administration. Pyridine dose levels for the UDS determination were set at 175, 350 and 700 mg kg(-1). Pyridine solutions in water were administered to mice 2 or 16 h prior to the scheduled sacrifice. The vehicle control group received water 16 h before sacrifice and the positive control group received 10 mg kg(-1) dimethylnitrosamine (DMN) 2 h before sacrifice. Pyridine did not significantly increase the UDS response in hepatocytes isolated from the treated animals, as measured by the incorporation of [3H]thymidine, using standard criteria for a negative response: less than zero mean net grains in repair (NG) and <20% of cells in repair (% IR; cells in repair have at least 5 NG). The vehicle control group and the low, mid- and high pyridine dose groups yielded less than -8.3 NG and < or =1% IR. The positive control group yielded a positive UDS response, with 10.8 NG and 62% IR. These results indicate that pyridine is non-genotoxic in B6C3F1 mouse liver using the UDS endpoint.     

The in vivo-in vitro hepatocyte assay for assessing DNA repair and DNA replication: studies in the CD-1 mouse

The in vivo-in vitro hepatocyte assay, a short-term test that is useful for detecting potential rodent hepatocarcinogens, has been extended to the CD-1 mouse. Male CD-1 mice, 50-100 days old, were dosed orally with N-nitrosodimethylamine (NDMA), trichloroethylene (TCE), 2-acetylaminofluorene (2-AAF), 4-acetylaminofluorene (4-AAF), phenobarbital (PB) or a vehicle. At 3, 16, 24 and 48 hr after dosing, hepatocytes were isolated by an in situ perfusion procedure, incubated in the presence of [3H]thymidine and fixed. Unscheduled as well as scheduled DNA synthesis was assessed by quantitative autoradiography. Unscheduled DNA synthesis represents DNA repair, while scheduled DNA synthesis (S phase) represents DNA replication. No mortality or morbidity was observed in the animals dosed in this study. The vehicles used, distilled water and corn oil, did not induce DNA repair or increase the percentage of hepatocytes in S phase at any of the time points examined. The higher dose of NDMA (10 mg/kg) induced DNA repair and increased S phase, whereas the lower dose (2 mg/kg) induced DNA repair but did not increase S phase. TCE (1000 mg/kg), PB (100 mg/kg) and 4-AAF (200 mg/kg) all markedly increased the percentage of hepatocytes in S phase (5-10 times the control) but all failed to induce DNA damage and repair. 2-AAF (200 mg/kg) caused a slight (twofold) increase in S phase but did not induce DNA repair. This assay appears useful for detecting potential CD-1 mouse hepatocarcinogens and, in addition, may provide information on the mode of action of known hepatocarcinogens.     

Evaluation of cultured, precision-cut rat liver slices as a model to study drug-induced liver apoptosis

The precision-cut liver slice culture model has been used widely to investigate drug metabolism and drug-induced necrosis. However, apoptosis, a key mediator of liver toxicity remains to be studied in this model. We evaluated apoptosis induced by thioacetamide (TAA) in rat liver slices, and in livers taken from TAA-treated rats as a control. Rat liver slices were treated with 50, 75 and 100 mM of TAA for 15 h. Histopathological examination of the liver slices revealed specific centrilobular localization of apoptotic hepatocytes at 75 mM but randomly distributed at 100 mM. Apoptosis in centrilobular hepatocytes was confirmed by appearance of cleavage products of caspase-3 and DNA fragmentation studied by TUNEL method. A concentration-dependent release of cytochrome c was observed in the slices, suggesting a role for mitochondria in the apoptosis triggered by TAA. The in vitro results were compared to the data obtained in male Sprague-Dawley rats given a single ip injection of 40 mg/kg TAA and sacrificed 1, 2, 3 and 6 h after dosing. Histopathological analyses showed specific centrilobular localization of apoptosis after 6 h treatment. Caspase-3 activation, DNA fragmentation and cytochrome c release were also observed in the liver of rats treated with TAA. Overall these data indicated that precision-cut liver slices provide a valuable in vitro system to study drug-induced liver apoptosis.     

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.     

Comparative Effects of Dieldrin on Hepatic Ploidy,Cell Proliferation,and Apoptosis in Rodent Liver

Dieldrin-induced hepatocarcinogenesis, which is seen only in the mouse, apparently occurs through a nongenotoxic mechanism. Previous studies have demonstrated that dieldrin induces hepatic DNA synthesis in mouse, but not rat liver. A number of nongenotoxic hepatocarcinogens have been shown to increase hepatocyte nuclear ploidy following acute and subchronic treatment in rodents, suggesting that an induction of hepatocyte DNA synthesis may occur without a concomitant increase in cell division. The current study examined the effects of dieldrin on changes in hepatocyte DNA synthesis, mitosis, apoptosis, and ploidy in mouse liver (the sensitive strain and target tissue for dieldrin-induced carcinogenicity) and the rat liver (an insensitive species). Male F344 rats and B6C3F1 mice were treated with 0, 1, 3, or 10 mg dieldrin/kg diet and were sampled after 7, 14, 28, or 90 d on diet. Liver from mice fed 10 mg dieldrin/kg diet exhibited significantly increased DNA synthesis and mitosis at 14, 28, or 90 d on diet. In rats, no increase in DNA synthesis or mitotic index was observed. The apoptotic index in liver of mice and rats did not change over the 90-d study period. Exposure of mice to only the highest dose of dieldrin produced a significant increase in octaploid (8N) hepatocytes and a decrease in diploid (2N) hepatocytes, which were restricted primarily to centrilobular hepatocytes, with the periportal region showing little or no change from control. No changes in hepatocyte nuclear ploidy were observed in the rat. This study demonstrates that exposure to high concentrations of dieldrin is accompanied by increased nuclear ploidy and mitosis in mouse, but not rat, liver. It is proposed that the observed increase in nuclear ploidy in the mouse may reflect an adaptive response to dieldrin exposure.     

In vivo genotoxic potential of microcystin-LR: a cyanobacterial toxin, investigated both by the unscheduled DNA synthesis (UDS) and the comet assays after intravenous administration

Microcystin-LR (MC-LR) is a toxin produced by freshwater cyanobacteria and is a potential threat to human health. MC-LR has been shown to be both a specific inhibitor of serine/threonine protein phosphatases PP1 and PP2A and a potent tumor promoter in rat liver. However, the genotoxic potential of MCs remains unclear. In this article, we investigated the ability of MC-LR to induce DNA damage on rat hepatocytes following intravenous (iv) administration by using two in vivo genotoxicity assays: the unscheduled DNA synthesis (UDS) and the comet assays. The UDS assay measures DNA synthesis induced from the excision repair of DNA damaged regions and the comet assay is a very sensitive technique for detecting various forms of DNA damage. After an exposure time of 2-4 h or 12-16 h and a dose ranging from 12.5 to 50 microg/kg bw, no DNA damage could be observed in both assays on rat hepatocytes following iv administration. These findings have been discussed and compared with recently published genotoxic results obtained in other organs from mice after oral and intraperitoneal treatments to better understand the mechanism of action of this toxin in relation with its cancerogenicity potential.     

Effects of ellagic acid by oral administration on distribution and metabolism of 2-aminofluorene in Sprague-Dawley rats

The effects of ellagic acid on the in vivo N-acetylation and metabolism of 2-aminofluorene (2-AF) were investigated in bladder, blood, colon, kidney, liver, feces and urine samples from male Sprague-Dawley rats. Major metabolites such as 1-OH-2-AAF, 8-OH-2-AAF and 9-OH-2-AAF were found in bladder tissues, 1-OH-2-AAF, 5-OH-2-AAF and 8-OH-2-AAF were found in blood samples, 1-OH-2-AAF, 3-OH-2-AAF, 5-OH-2-AAF, 8-OH-2-AAF and 9-OH-2-AAF were found in colon tissues, 1-OH-2-AAF, 3-OH-2-AAF and 9-OH-2-AAF were found in kidney tissues, 1-OH-2-AAF, 3-OH-2-AAF and 8-OH-2-AAF were found in liver tissues, 1-OH-2-AAF, 3-OH-2-AAF, 5-OH-2-AAF and 8-OH-2-AAF were found in feces samples and 1-OH-2-AAF, 3-OH-2-AAF, 5-OH-2-AAF and 8-OH-2-AAF were also found in urine samples after rats had been orally treated with 2-AF (50 mg/kg) for 24 h. Pretreatment of male rats with ellagic acid (10 mg/kg) 24 h prior to the administration of 2-AF (50 mg/kg) resulted in absence of 8-OH-2-AAF in bladder tissues, and there were significant decreases of 8-OH-2-AAF in blood and urine samples. In blood samples, amounts of 2-AAF and 8-OH-2-AAF were significantly decreased; in colon tissues, amounts of 2-AF, 1-OH-2-AAF and 3-OH-2-AAF, in liver tissues, amounts of 2-AAF, 1-OH-2-AAF and 3-OH-2-AAF, and in urine samples, amounts of 2-AF and 8-OH-2-AAF were significantly decreased in 24-h ellagic acid (EA)-treated rats before 2-AF was added to the diet. However, significantly increased 1-OH-2-AAF concentrations were found in urine samples in 24-h EA-treated rats before 2-AF was administered. In the EA and 2-AF rats, in the same time treated groups, bladder, colon and liver tissues, and feces and urine samples showed significant differences when compared to the ones without EA co-treatment. We saw significant decreases of the amounts of 2-AF and 1-OH-2-AAF in colon tissues. The feces samples showed increased amounts of 2-AAF in EA- and in 2-AF- treated rats in the same time groups, but urine samples showed a decreased amount of 8-OH-2-AAF in both EA-treated groups. The total amounts of 2-AF metabolites in bladder, blood, kidney and liver tissues showed significant difference between control and the group which was EA-treated 24 h before 2-AF was added. The total amounts of 2-AF metabolites in the liver, feces and urine showed significant decreases between control and EA-treated at the same time with 2-AF groups. This is the first report of EA affecting the N-acetylation and metabolism of 2-AF in rat tissues in vivo.     

Dose-dependency of 2-acetylaminofluorene binding to liver DNA and hemoglobin in mice and rats

The carcinogenic activity of 2-acetylaminofluorene (2-AAF) in mice and rats has been accurately described so that it is a suitable archetypical chemical for investigations of parameters of cross-species variation. The binding of orally administered [9-¹⁴C]2-AAF to liver DNA in mice and rats was dose related in a linear manner between 1 to 100 μmol/kg body wt. By Sephadex LH-20 chromatography, it was determined that the formation of 2-AAF adducts over the dose range studied was both qualitatively and quantitatively similar. Hepatic DNA binding was greater in rats compared to mice. No sex difference was observed in the DNA binding in rats so that the higher incidence of liver cancer in male compared to female rats could not be explained by a greater amount of DNA binding in males. The binding of orally administerd [9-¹⁴C]2-AAF to hemoglobin in mice and rats was dose related in a linear manner between 0.1 to 100 μmol/kg. The results of 2-AAF binding to DNA and hemoglobin would indicate that for an oral dose of 2-AAF, the rate of formation of reactive metabolites was similar between 0.1 and 100 μmol/kg body wt. However, the sevenfold greater DNA binding and 2.5-fold greater hemoglobin binding in rats compared to mice were more than an order of magnitude less than 246-fold greater carcinogenicity of 2-AAF in rat liver. The results of the study supported a pharmacokinetic model that related the metabolism of 2-AAF to reactive metabolites to the binding of the metabolites to DNA and hemoglobin. The results did not support a relationship of macromolecular binding to carcinogenic potency.     

DISTRIBUTION AND METABOLISM OF (5-HYDROXYMETHYL)FURFURAL IN MALE F344 RATS AND B6C3F1 MICE AFTER ORAL ADMINISTRATION

(5-Hydroxymethyl)furfural (HMF), a heat-induced decomposition product of hexoses, is present in food and drink. Recent reports have shown HMF to be an in vitro mutagen after sulfate conjugation and to be a promoter as well as a weak initiator of colonic aberrant foci in rats. In order to investigate the metabolic activation further and to provide information for HMF toxicology studies, the disposition of [14C]-HMF has been investigated in male F344 rats and B6C3F1 mice following po administration of either 5, 10, 100, or 500 mg/kg. Tissue distribution results indicated that absorption of HMF was rapid in male rats and mice and that tissue concentrations in male mice at the earliest time point are not linearly proportional to dose. Excretion was primarily via the urine in both, with 60-80% of the administered dose excreted by this route in 48 h. Tissue/blood ratios of HMF-derived radioactivity were greater than 1 for liver and kidney. Three metabolites were identified and quantitated in urine. Formation of one of the metabolites, N-(5-hydroxymethyl-2-furoyl)glycine, was inversely proportional to dose in rats but not mice. None of the metabolites were sulfate conjugates nor likely to be formed from sulfate conjugates. There were relatively low levels of nonextractable radioactivity in liver, kidney, and intestines, indicating that some reactive intermediate(s) may be formed.     

Effects of oral administration of berberine on distribution and metabolism of 2-aminofluorene in Sprague-Dawley rats

The effects of berberine on the in vivo N-acetylation and metabolism of 2-aminofluorene (2-AF) in bladder, blood, colon, kidney, liver, feces and urine samples and brain tissues (cerebrum, cerebellum and pineal gland) of male Sprague-Dawley rats were investigated. Major metabolites, such as 1-OH-2-AAF, 3-OH-2-AAF, 8-OH-2-AAF and 9-OH-2-AAF were found in bladder tissues, 1-OH-2-AAF, 5-OH-2-AAF and 8-OH-2-AAF were found in blood samples, 1-OH-2-AAF, 3-OH-2-AAF, 5-OH-2-AAF, 8-OH-2-AAF and 9-OH-2-AAF were found in colon tissues, 1-OH-2-AAF, 3-OH-2-AAF and 9-OH-2-AAF were found in kidney tissues, 1-OH-2-AAF, 3-OH-2-AAF and 8-OH-2-AAF were found in liver tissues, 1-OH-2-AAF, 3-OH-2-AAF, 5-OH-2-AAF, 7-OH-2-AAF, 8-OH-2-AA and 9-OH-2-AAF were found in feces samples and 1-OH-2-AAF, 3-OH-2-AAF, 5-OH-2-AAF, 7-OH-2-AAF, 8-OH-2-AA and 9-OH-2-AAF were also found in urine samples, 1-OH-2-AAF, 3-OH-2-AAF and 8-OH-2-AAF were found in cerebrum tissues, 1-OH-2-AAF, 3-OH-2-AAF and 7-OH-2-AAF were found in cerebellum tissues. In the control group, however, only 2-AF and 2-AAF were found in pineal gland after rats had been orally treated with 2-AF (50 mg/kg) for 24 h. Pre-treatment of male rats with berberine (40 mg/kg) 24 h prior to the administration of 2-AF (50 mg/kg), as well as the co-administration of berberine and 2-AF led to a decrease in the amounts of 3-OH-2-AAF and an increase in the amounts of 8-OH-2-AAF in bladder tissues. In blood samples, there were significant decreases of 2-AF, 2-AAF, 1-OH-2-AAF and 8-OH-2-AAF, after rats were pre-treated with berberine for 24 h before the addition of 2-AF. However, co-administration of berberine and 2-AF led to an increase in the amounts of 5-OH-2-AAF. In colon tissues, there were significant decreases of 2-AF, 2-AAF, 1-OH-2-AAF and 8-OH-2-AAF in colon samples after rats were treated with berberine for 24 h before the addition of 2-AF. 2-AF, 1-OH-2-AAF, 3-OH-2-AAF and 9-OH-2-AAF levels were significantly different between control and the group treated with berberine and 2-AF at the same time. In kidney tissues, significant decreases of 2-AF and 2-AAF and of 3-OH-2-AAF were observed after rats were treated with both compounds separately and simultaneously. However, 24 h berberine pre-treatment followed by addition of 2-AF led to significant increase of 9-IH-2-AAF. In liver tissues, there were significant decreases of 2-AAF and 1-OH-2-AAF, after co-administration of berberine and 2-AF. The amounts of 2-AAF, 1-OH-2-AAF and 3-OH-2-AAF were significantly different between the control and the group pretreated with berberine 24 h before the addition of 2-AF. In the feces samples, there were significant decreases of 2-AAF, 3-OH-2-AAF, 7-OH-2-AAF, 8-OH-2-AAF and 9-OH-2-AAF after co-administration of berberine and 2-AF. However, the berberine pre-treatment followed by addition of 2-AF led to a significant increase of 2-AF, 2-AAF and 1-OH-2-AAF levels. In urine samples, there were significant differences of 2-AF, 2-AAF, 1-OH-2-AAF, 3-OH-2-AAF, 5-OH-2-AAF, 8-OH-2-AAF and 9-OH-2-AAF after the co-treatment. However, berberine treatment followed by 2-AF led to significant differences in 1-OH-2-AAF and 5-OH-2-AAF levels. In the cerebrum samples, there were significant differences in 1-OH-2-AAF and 8-OH-2-AAF after both berberine co-treatment and pre-treatment. In cerebellum samples, there were also significant differences in the 1-OH-2-AAF and 3-OH-2-AAF levels after both co- and pretreatment. In pineal gland samples, there were significant differences in 2-AAF levels after co-treatment with berberine and 2-AF and 1-OH-2-AAF was also found in both groups. However, berberine pre-treatment followed by 2-AF led to different levels of 2-AF and 2-AAF, but not of 3-OH-2-AAF.     

Comparative effects of dieldrin on hepatic ploidy, cell proliferation, and apoptosis in rodent liver

Dieldrin-induced hepatocarcinogenesis, which is seen only in the mouse, apparently occurs through a nongenotoxic mechanism. Previous studies have demonstrated that dieldrin induces hepatic DNA synthesis in mouse, but not rat liver. A number of nongenotoxic hepatocarcinogens have been shown to increase hepatocyte nuclear ploidy following acute and subchronic treatment in rodents, suggesting that an induction of hepatocyte DNA synthesis may occur without a concomitant increase in cell division. The current study examined the effects of dieldrin on changes in hepatocyte DNA synthesis, mitosis, apoptosis, and ploidy in mouse liver (the sensitive strain and target tissue for dieldrin-induced carcinogenicity) and the rat liver (an insensitive species). Male F344 rats and B6C3F1 mice were treated with 0, 1, 3, or 10 mg dieldrin/kg diet and were sampled after 7, 14, 28, or 90 d on diet. Liver from mice fed 10 mg dieldrin/kg diet exhibited significantly increased DNA synthesis and mitosis at 14, 28, or 90 d on diet. In rats, no increase in DNA synthesis or mitotic index was observed. The apoptotic index in liver of mice and rats did not change over the 90-d study period. Exposure of mice to only the highest dose of dieldrin produced a significant increase in octaploid (8N) hepatocytes and a decrease in diploid (2N) hepatocytes, which were restricted primarily to centrilobular hepatocytes, with the periportal region showing little or no change from control. No changes in hepatocyte nuclear ploidy were observed in the rat. This study demonstrates that exposure to high concentrations of dieldrin is accompanied by increased nuclear ploidy and mitosis in mouse, but not rat, liver. It is proposed that the observed increase in nuclear ploidy in the mouse may reflect an adaptive response to dieldrin exposure.     

Toxicology and carcinogenesis studies of riddelliine (CAS No. 23246-96-0) in F344/N rats and B6C3F1 mice (gavage studies)

Riddelliine belongs to a class of toxic pyrrolizidine alkaloids and is isolated from plants of the genera Crotalaria, Amsinckia, and Senecio that grow in the western United States. Cattle, horses, and sheep that ingest these plants succumb to their toxic effects. Riddelliine residues have been found in meat, milk, and honey, and the plants may contaminate human food sources. Riddelliine was nominated for study by the Food and Drug Administration because of its potential for human exposure and its economic impact on the livestock industry and because the toxicity of other pyrrolizidine alkaloids suggests riddelliine may be carcinogenic. Male and female F344/N rats and B6C3F1 mice received riddelliine (approximately 92% pure) by gavage. Female rats and male and female mice were dosed for 2 years; due to high mortality, the study in male rats was terminated at week 72. In vitro genetic toxicology studies were conducted in Salmonella typhimurium and in cultured Chinese hamster ovary (CHO) cells. In addition, riddelliine was evaluated in vivo for induction of micronuclei in mouse bone marrow and peripheral blood erythrocytes and for induction of S-phase DNA synthesis and unscheduled DNA synthesis in the liver of rats and mice. Riddelliine-induced DNA adduct levels were determined in liver tissue obtained from female rats admininstered riddelliine for 3 or 6 months. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 0 or 1 mg riddelliine/kg body weight in sodium phosphate buffer by gavage 5 days per week; additional groups of 50 female rats received 0.01, 0.033, 0.1, or 0.33 mg/kg. A wide dose range was used in female rats to better characterize the dose-response curve. Females were dosed for 105 weeks; due to high mortality, male rats were terminated at week 72. All but three 1 mg/kg males died before week 70, and all 1 mg/kg females died before week 97. Mean body weights of 1 mg/kg males and females were less than those of the vehicle controls throughout most of the study. The only clinical finding related to riddelliine administration was a general debilitation of the animals prior to death. Hemangiosarcomas were present in the liver of 86% of males and 76% of females in the 1 mg/kg groups, and this neoplasm was considered the cause of the large number of early deaths in these groups. The incidences of hepatocellular adenoma and mononuclear cell leukemia in 1 mg/kg males and females were significantly increased. Nonneoplastic lesions related to riddelliine treatment occurred in the liver and kidney of males and females. Analyses of liver tissue from female rats treated with riddelliine for 3 or 6 months yielded eight DNA adducts; these were the same as DNA adducts formed in vitro by the metabolism of riddelliine by human liver microsomes in the presence of calf thymus DNA. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered riddelliine in sodium phosphate buffer by gavage at doses of 0 or 3 mg/kg, 5 days per week, for 105 weeks; additional groups of 50 male mice received 0.1, 0.3, or 1 mg/kg for 105 weeks. A wide dose range was used in male mice to better characterize the dose-response curve. Survival of males and females administered 3 mg/kg was significantly less than that of the vehicle controls. Mean body weights of 3 mg/kg mice were less than those of the vehicle controls throughout most of the study. Hemangiosarcomas of the liver were present in 62% of males in the 3 mg/kg group. The incidences of hepatocellular neoplasms occurred with negative trends in male mice and were significantly decreased in 3 mg/kg females. The incidences of alveolar/bronchiolar neoplasms in 3 mg/kg females were significantly increased. Nonneoplastic lesions related to riddelliine administration occurred in the liver and kidney of males and females and in the lung and arteries (multiple tissues) of females. GENETIC TOXICOLOGY: Riddelliine was mutagenic in S. typhimurium strain TA100 with, but not without, S9 activation; no significant mutagenic activity was detected in strain TA98 or TA1535,ed in strain TA98 or TA1535, with or without S9. A small, dose-related increase in mutant colonies seen in strain TA97 with S9 was judged to be equivocal. Riddelliine induced sister chromatid exchanges in cultured CHO cells with and without S9. Chromosomal aberrations were induced in CHO cells only in the presence of S9. Following 4 or 13 weeks of daily gavage treatment with riddelliine, no increases in the frequency of micronucleated erythrocytes were noted in the peripheral blood of male or female B6C3F1 mice. Use of a single intraperitoneal injection protocol, however, produced a small but significant increase in the frequency of micronucleated eryth-rocytes in peripheral blood of male Swiss mice 48 hours after injection; bone marrow analysis 24 hours after injection demonstrated a small but insignificant increase in the frequency of micronuclei. Unscheduled DNA synthesis was detected in cultured hepatocytes from male and female rats and mice following 5 or 30 days of riddelliine treatment by gavage. In addition, an S-phase DNA synthesis was observed in cultured hepatocytes of male and female rats treated for either time period. CONCLUSIONS: Under the conditions of these studies, there was clear evidence of carcinogenic activity of riddelliine in male and female F344/N rats based primarily on increased incidences of hemangiosarcoma in the liver. The increased incidences of hepatocellular adenoma and mononuclear cell leukemia in male and female rats were also considered to be treatment related. There was clear evidence of carcinogenic activity of riddelliine in male B6C3F1 mice based on increased incidences of hemangiosarcoma in the liver. There was clear evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of alveolar/bronchiolar neoplasms. Administration of riddelliine by gavage resulted in nonneoplastic lesions in the liver and kidney of male and female rats; the liver and kidney of male and female mice; and the lung and arteries (multiple tissues) of female mice. Decreased incidences of hepatocellular neoplasms in male and female mice were related to riddelliine administration.     

Subchronic Effects of Dieldrin and Phenobarbital on Hepatic DNA Synthesis in Mice and Rats

Dieldrin, an organochlorine pesticide, has been shown to behepatocarcinogenic in mice but not rats. Phenobarbital, in contrast,induces hepatic tumors in both mice and rats. Previous studieshave shown that acute dietary exposure of rats or mice to eitherdieldrin or phenobarbital produces several liver changes, includingcentrilobular hypertrophy, induction of hepatic cytochrome P450,and increased liver weight. The present study examined the subchroniceffect of dieldrin (0.1, 1.0, 3.0, 10.0 mg dieldrin/kg diet)and phenobarbital (10, 50, 100, 500 mg phenobarbital/kg diet)on the induction of hepatic DNA synthesis and hepatocyte lethalityin male B6C3F1 mice and male F344 rats. Eight-week-old animalswere treated as above and evaluated for hepatic DNA synthesisafter 7, 14, 21, 28, and 90 days of continual treatment to dieldrinor phenobarbital. Maximal induction of hepatic DNA synthesisin mice was seen at the 14-, 21-, and 28-day sampling times.In rats, no significant increase in hepatic DNA synthesis orhepatocyte lethality was observed at any dose of dieldrin investigated.Phenobarbital produced a significant increase in hepatic DNAsynthesis in both rat and mouse liver following 7 days of treatment.The induction of DNA synthesis in rat liver was transient, withthe labeling index returning to control levels by 14 days oftreatment. In contrast, mice treated with phenobarbital showeda significant increase in hepatic DNA synthesis throughout thetreatment. In both mice and rats, dieldrin and phenobarbitalinduced hepatic DNA synthesis selectively in the centrilobularregion of the hepatic lobule. The lack of an increase in serumenzymes indicative of hepatic damage and the absence of liverhistopathology in mice or rats fed dieldrin or phenobarbitalindicate that the induction of DNA synthesis was not mediatedby a cytolethal, compensatory hyperplastic response, suggestinga mitogenic mechanism. Therefore, the species-specific inductionof hepatic DNA synthesis by either dieldrin or phenobarbitalcorrelated with the previously observed species-specific inductionof hepatic cancer by these two compounds.     

Distribution and metabolism of (5-hydroxymethyl)furfural in male F344 rats and B6C3F1 mice after oral administration.

(5-Hydroxymethyl)furfural (HMF), a heat-induced decomposition product of hexoses, is present in food and drink. Recent reports have shown HMF to be an in vitro mutagen after sulfate conjugation and to be a promoter as well as a weak initiator of colonic aberrant foci in rats. In order to investigate the metabolic activation further and to provide information for HMF toxicology studies, the disposition of [14C]-HMF has been investigated in male F344 rats and B6C3F1 mice following po administration of either 5, 10, 100, or 500 mg/kg. Tissue distribution results indicated that absorption of HMF was rapid in male rats and mice and that tissue concentrations in male mice at the earliest time point are not linearly proportional to dose. Excretion was primarily via the urine in both, with 60-80% of the administered dose excreted by this route in 48 h. Tissue/blood ratios of HMF-derived radioactivity were greater than 1 for liver and kidney. Three metabolites were identified and quantitated in urine. Formation of one of the metabolites, N-(5-hydroxymethyl-2-furoyl)glycine, was inversely proportional to dose in rats but not mice. None of the metabolites were sulfate conjugates nor likely to be formed from sulfate conjugates. There were relatively low levels of nonextractable radioactivity in liver, kidney, and intestines, indicating that some reactive intermediate(s) may be formed.     

A dual-labeling method to quantify unscheduled DNA synthesis in primary cells

Unscheduled DNA synthesis (UDS) has been used as an endpoint for measuring DNA damage in vitro and in vivo. Determination of UDS is regarded as a reliable genotoxicity assay by regulatory agencies including US FDA and EPA. In this study, we have developed an improved UDS assay to detect DNA damage and repair processes upon chemical exposure. We utilized a dual-labeling procedure in which fluorescent DAPI stained nuclei of live cells and [(3)H]thymidine labeled cells undergoing new DNA synthesis. The occurrence of UDS in cells was quantified by either manual nucleus counting or net intensity approaches. This assay was validated by testing known genotoxic compounds 2-acetylaminofluorene (2-AAF), ethylmethanesulfonate (EMS), and N-nitrosodimethylamine (NDL) in primary rat hepatocytes as well as in confluent human mammary epithelial cells. In addition, fluorescent labeling of nuclei DNA helped to distinguish apoptotic cells from non-apoptotic cells. Chemical effects on cell functions were also examined by conducting the cytotoxicity assay along with the UDS assay. To conclude, the dual-labeling UDS assay offers advantages of reduced subjective bias, increasing sensitivity and reproducibility. The assay is suitable for testing compounds in higher capacity format with much less compound needed.     

Toxicity of aflatoxin B1 in rat and mouse hepatocytes in vivo and in vitro

The reported LD50 for the adult, male Fisher rat is 1.2 mg aflatoxin B1/kg body weight (i.p.); we have found that male C57BL/6 mice survive single doses of aflatoxin B1 as high as 60 mg/kg (i.p.). We have demonstrated a 1000-fold greater LC50 of aflatoxin B1 for primary mouse liver cell cultures from C57BL/6 male mice (3 X 10(-5) M) than for primary liver cells from F344 male rats (3 X 10(-8) M). In 4 h of exposure to a non-toxic dose (1 X 10(-9) M) of [3H]aflatoxin B1, cultured rat liver cells accumulated up to 5-fold higher concentrations of 3H label than did mouse liver cells. The difference in cell-associated counts was due largely to higher levels of aflatoxin B1 metabolites bound to macromolecules in the rat cells.     

Trichloroacetate stimulation of liver DNA synthesis in male and female mice

Male and female B6C3F1 mice were given trichloroacetate (TCA) by gavage for 11 days. Livers from untreated male and female mice were unremarkable by histopathologic examination. In livers from mice receiving 1000 mg/kg body weight, the centrolobular hepatic cords showed slight changes, which included increased eosinophilic staining and rare apoptosis. Areas in the intermediate zone were noted where the architecture of the liver hepatic cords was subtly changed. The changes in cord architecture seemed to define nodular areas where cellular proliferation in animals treated with TCA had occurred. No histopathologic differences were noted between the livers of treated or control, male and female animals. Mitosis and DNA synthesis were examined using incorporation of ³H]thymidine into liver cells. ³H]Thymidine incorporation into extracted liver DNA of animals receiving TCA was significantly increased over controls in all treatment groups. Autoradiographic examination of liver sections showed that the incorporation of label in control animals was predominantly in peri-sinusoidal cells, whereas the majority of radiolabel incorporation in TCA-treated animals was found in intermediate zone cells that appeared to be mature hepatocytes. No outstanding differences in the distribution of radiolabel in the liver sections from male or female mice were noted. When incorporation of ³H]thymidine was quantified by enumeration of labeled liver cells following autoradiography, incorporation of the radiolabel into hepatocytes increased with the dose of TCA given but there was no increase in radiolabel in peri-sinusoidal cells. Increased mitotic figures in intermediate zone cells resembling mature hepatocytes were noted in all mice treated with TCA. These results suggest that increased DNA synthesis and mitosis may contribute tumorigenesis by TCA.