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.     

Lack of effect of furfural on unscheduled DNA synthesis in the in vivo rat and mouse hepatocyte DNA repair assays and in precision-cut human liver slices.

The ability of furfural to induce unscheduled DNA synthesis (UDS) in hepatocytes of male and female B6C3F(1) mice and male F344 rats after in vivo administration and in vitro in precision-cut human liver slices has been studied. Preliminary toxicity studies established the maximum tolerated dose (MTD) of furfural to be 320 and 50 mg/kg in the mouse and rat, respectively. Furfural was dosed by gavage at levels of 0 (control), 50, 175 and 320 mg/kg to male and female mice and 0, 5, 16.7 and 50 mg/kg to male rats. Hepatocytes were isolated by liver perfusion either 2-4 h or 12-16 h after treatment, cultured in medium containing [3H]thymidine for 4 h and assessed for UDS by grain counting of autoradiographs. Furfural treatment did not produce any statistically significant increase or any dose-related effects on UDS in mouse and rat hepatocytes either 2-4 h or 12-16 h after dosing. In contrast, UDS was markedly induced in mice and rats 2-4 h after treatment with 20 mg/kg dimethylnitrosamine and 12-16 h after treatment of mice and rats with 200 mg/kg o-aminoazotoluene and 50 mg/kg 2-acetylaminofluorene (2-AAF), respectively. Precision-cut human liver slices from four donors were cultured for 24 h in medium containing [3H]thymidine and 0-10 mM furfural. Small increases in the net grain count (i.e. nuclear grain count less mean cytoplasmic grain count) observed with 2-10 mM furfural were not due to any increase in the nuclear grain count. Rather, it was the result of concentration-dependent decreases in the mean cytoplasmic grain counts and to a lesser extent in nuclear grain counts, due to furfural-induced cytotoxicity. In contrast, marked increases in UDS (both net grain and nuclear grain counts) were observed in human liver slices treated with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 mM aflatoxin B(1) and 0.005 and 0.05 mM 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. This study demonstrates that furfural does not induce UDS in the hepatocytes of male and female B6C3F(1) mice and male F344 rats after oral treatment at doses up to the MTDs. Moreover, human liver slice studies suggest that furfural is also not a genotoxic agent in human liver.     

Effect of di(2-ethylhexyl) phthalate on DNA repair and lipid peroxidation in rat hepatocytes and on metabolic cooperation in Chinese hamster V-79 cells

Experiments were conducted to test the hypothesis that the hepatocarcinogenicity of di(2-ethylhexyl) phthalate (DEHP) is due to its ability to produce DNA damage, either directly or as a result of the proliferation of peroxisomes and accompanying increased production of H2O2 and other DNA--damaging oxygen radicals induced by sustained exposure to the plasticizer. DNA repair, as assessed by the autoradiographic measurement of unscheduled DNA synthesis (UDS), was not observed in primary rat hepatocytes exposed in vitro to 10(-5)-10(-2) M DEHP or in vivo by a single gavage dose of 5 g DEHP/kg body weight administered 2, 15, or 24 h prior to the isolation of hepatocytes. Thus, DEHP does not appear to directly produce repairable DNA damage in rat hepatocytes. Sustained feeding of DEHP at a dietary concentration of 2% led to a marked proliferation of peroxisomes in the liver after 4 wk. Additional administration of a single gavage dose of 5 g DEHP/kg body weight to animals fed the 2% diet for 4 or 8 wk, as well as to 4-wk-fed animals that were also pretreated with 3-amino-1,2,4-triazole to inhibit endogenous catalase activity, did not induce any detectable DNA repair in hepatocytes isolated 15 h following the single gavage dose of DEHP. Lipid peroxidation measured in the 9000 X g supernatant of livers from animals treated with a single dose of 5 g DEHP/kg body weight or the 2% DEHP diet for 6 wk plus a single dose of 5 g/kg body weight did not differ from controls. These findings suggest that DEHP does not elicit DNA damage or lipid peroxidation in liver consequent to the proliferation of peroxisomes resulting from prolonged administration. In addition, at noncytotoxic concentrations DEHP failed to produce a positive response in the Chinese hamster V-79 metabolic cooperation assay for tumor promoters.     

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.     

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.     

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.     

Minimal effect of ketoconazole on cyclosporine (SangCyA) oral absorption and first-pass metabolism in rats: evidence of a significant vehicle effect on SangCyA absorption

The current work evaluated the effect of the CYP3A inhibitor ketoconazole on the oral absorption and first-pass metabolism of cyclosporine administered as the SangCyA formulation. Groups of 6 male Sprague-Dawley rats were administered SangCyA (5 and 15 mg/kg) by oral gavage alone and with ketoconazole (30 mg/kg). Blood cyclosporine levels were measured over 6 h, encompassing the cyclosporine absorption window. A significant vehicle effect on SangCyA absorption was observed. Comparing a 15 mg/kg dose, cyclosporine C(max) (mean+/-SD 1.12+/-0.16 microg/ml) and AUC(0-6) (5.34+/-0.71 microg h/ml) were 50% lower when propylene glycol was used as gavage vehicle instead of saline (2.19+/-0.94 microg/ml and 9.52+/-2.52 microg h/ml, respectively). Coefficients-of-variation for these parameters were halved in the propylene glycol vehicle however T(max) was unaffected. Ketoconazole increased cyclosporine C(max) and AUC(0-6) by 50-60%, regardless of the vehicle or the cyclosporine dose, without altering T(max) (2-3 h). The small effect of ketoconazole suggests that CYP3A-mediated intestinal and first-pass hepatic metabolism are minor determinants of cyclosporine oral bioavailability in rats.     

The effect of route, vehicle, and divided doses on the pharmacokinetics of chlorpyrifos and its metabolite trichloropyridinol in neonatal Sprague-Dawley rats.

There is a paucity of data on neonatal systemic exposure using different dosing paradigms. Male CD (Sprague-Dawley derived) rats at postnatal day (PND) 5 were dosed with chlorpyrifos (CPF, 1 mg/kg) using different routes of exposure, vehicles, and single versus divided doses. Blood concentrations of CPF and its primary metabolite, trichloropyridinol, were measured at multiple times through 24 h. Groups included were single gavage bolus versus divided gavage doses in corn oil (one vs. three times in 24h), single gavage bolus versus divided gavage doses in rat milk, and sc administration in dimethyl sulfoxide (DMSO). These data were compared with lactational exposure of PND 5 pups from dams exposed to CPF in the diet at 5 mg/kg/day for 4 weeks or published data from dams exposed to daily gavage with CPF at 5 mg/kg/day. Maternal blood CPF levels were an order of magnitude lower from dietary exposure than gavage (1.1 vs. 14.8 ng/g), and blood CPF levels in PND 5 pups that nursed dietary-exposed or gavage-exposed dams were below the limit of detection. Single gavage doses of 1 mg/kg CPF in corn oil vehicle in pups resulted in CPF blood levels of 49 ng/g and in milk vehicle about 9 ng/g. Divided doses led to lower peak CPF levels. A bolus dose of 1 mg/kg CPF in DMSO administered sc appeared to have substantially altered pharmacokinetics from orally administered CPF. To be meaningful for risk assessment, neonatal studies require attention to the exposure scenario, since route, vehicle, dose, and frequency of administration result in different systemic exposure to the test chemical and its metabolites.     

Lack of effect of coumarin on the formation of micronuclei in an in vivo mouse micronucleus assay.

Coumarin was tested for its potential to cause genotoxic effects in mouse bone marrow cells using an in vivo micronucleus assay. Male and female Swiss mice were administered a single oral dose of coumarin at 50, 100 or 200 mg/kg by gavage in corn oil vehicle. Control animals received only the vehicle. Groups of male mice were also administered mitomycin C at 0.75 mg/kg and served as positive controls. At 24 h after treatment, mice from all dose levels, and at 48 h after treatment, mice from the high dose level only were sacrificed. Bone marrow cells were collected and assayed for the presence of micronuclei. Coumarin did not cause any increase in the incidence of micronucleated polychromatic erythrocytes in male or female mice at any of the dose levels, the positive control mitomycin C produced a significant increase. There was no evidence of coumarin or mitomycin C treatment related cytotoxicity to bone marrow cells. The results of this study demonstrate that coumarin is negative in the mouse in vivo micronucleus assay.     

Induction by pyridine of cytochrome P450IIE1 and xenobiotic metabolism in rat lung and liver.

Pyridine has been shown to induce hepatic microsomal cytochrome P450IIE1 and dependent activities. In the present study a single dose of pyridine (200 mg/kg body weight i.p.) induced cytochrome P450IIE1 in both the liver and lung. Increases were observed in p-nitrophenol hydroxylation, ethoxyresorufin deethylation and N-nitrosodimethylamine metabolism in both tissues. Pyridine appears to be an excellent inducer of this isozyme in the lung and may be useful in studying xenobiotic metabolizing activities in this organ which are dependent upon this isozyme.     

Induction of unscheduled DNA synthesis in rat and hamster hepatocytes by cooked food mutagens

The genotoxicity of the cooked-food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was studied by monitoring the induction of DNA repair (unscheduled DNA synthesis; UDS) in primary cultures of rodent hepatocytes. The hepatocytes were derived from male Sprague-Dawley rats or Syrian hamsters by collagenase perfusion and the cells were cultured for 4 hr before being exposed to various concentrations of the mutagens. DNA repair was determined by measuring incorporation of [3H]thymidine into extracted DNA over 17 hr using beta-scintillation counting. Dose-related increases in UDS were clearly seen in hamster hepatocytes treated with MeIQ, IQ and the positive control 2-acetylaminofluorene (AAF), and a weak response was induced by MeIQx and Trp-P-1. In the rat hepatocytes only MeIQ and AAF gave clear positive responses. Furthermore it was noted that all the mutagens displayed a more pronounced UDS response in hamster hepatocytes than in rat cells. Studies of the activation of MeIQ by hepatocytes to a bacterial mutagen suggest that this difference is probably a consequence of the greater capacity of hamster cells to activate the mutagens to genotoxic metabolites.     

Lack of Effects of Ciprofloxacin and the Topoisomerase II Inhibitors,m-AMSA and Nalidixic Acid, on DNA Repair in Cultured Rat Liver Cells

Several quinolone antibiotics, including ciprofloxacin, have been reported to elicit autoradiographic unscheduled DNA synthesis (UDS) in cultured rat hepatocytes. In the present investigation, ciprofloxacin (CF), at 250–1500 μM, produced autoradiographic UDS in cultured rat hepatocytes, whereas neither the quinolone nalidixic acid norm-AMSA, both topoisomerase II inhibitors, produced autoradiographic UDS. CF also reduced cytoplasmic [³H]thymidine levels ([³H]TdR) relative to control at 250–1500 μMand concomitantly increased nuclear grain counts accounting for most of the net increase yielding positive UDS values. To obtain definitive information on whether the positive UDS observed with CF was due to DNA repair, DNA repair synthesis was measured in parental DNA separated from newly replicated DNA using a bromodeoxyuridine incorporation density gradient method. This method was used to measure DNA repair synthesis in parental DNA of both replicating rat liver epithelial cells (ARL-18) and nonproliferating rat hepatocytes in primary culture. Primary hepatocytes exposed to CF from 250 to 1500 μMdid not express DNA repair synthesis in parental DNA isolated by density gradient centrifugation but rather exhibited a concentration-related decrease in the level of [³H]TdR associated with DNA. In rat liver epithelial (ARL-18) cells, CF from 250 to 500 μMlikewise did not elicit DNA repair synthesis and also caused a concentration-related decrease in the level of [³H]TdR associated with parental DNA. In contrast, in both cell types a substantial level of repair synthesis occurred in parental DNA as a result of exposure to 2-acetylaminofluorene, a DNA-reactive carcinogen, and in hepatocytes a similar finding was made for the drug hydralazine. Also, after induction of DNA repair in hepatocytes by ultraviolet light, the DNA polymerase α inhibitor aphidicolin almost completely abolished repair synthesis, whereas CF had a negligible effect on the inhibition of repair relative to control. These results indicate that CF did not elicit authentic DNA repair and also did not inhibit DNA repair synthesis. The fact that CF elicited autoradiographic UDS and that the topoisomerase II inhibitorsm-AMSA and nalidixic acid did not indicates that effects on topoisomerase II are not the basis for the positive UDS result with CF as has been hypothesized in the past.     

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.     

Disposition of [14C]N,N-dimethyl-p-toluidine in F344 rats and B6C3F1 mice

N,N-Dimethyl-p-toluidine (DMPT) is used as a polymerization accelerator, in industrial glues, and as an intermediate in dye and pesticide synthesis. There is potential for human exposure to DMPT. The disposition of oral and intravenous (i.v.) doses of [14C]DMPT in F344 rats and B6C3F1 mice was investigated. A single i.v. (2.5 mg/kg) or oral (2.5, 25, or 250 mg/kg) dose of [14C]DMPT (1-25 microCi) was administered in an aqueous vehicle to male rats and mice. The 25-mg/kg oral dose was administered to females to investigate possible gender differences in disposition. However, no striking gender differences were observed. Since toxicity studies conducted elsewhere used a corn oil vehicle, the 250-mg/kg oral dose also was administered in corn oil to male rats; disposition was not dependent on vehicle. Excreta (through 24 h) and tissues collected at sacrifice were analyzed for total radioactivity. Dose-dependent differences in toxicity and disposition were observed. Toxicity at the 250-mg/kg oral dose to male mice was consistent with acute renal failure. At the same dose, male rats exhibited clinical signs of toxicity through 12 h but were clinically normal by 24 h. At lower oral doses, [14C]DMPT-derived radioactivity was well absorbed and rapidly excreted, primarily in urine.     

Oncogenicity evaluation of resveratrol in p53(+/-) (p53 knockout) mice.

A six-month study was conducted in p53(+/-) mice to evaluate the possible oncogenicity of resveratrol (3,5,4'-trihydroxy-trans-stilbene), a cancer chemopreventive agent present in grapes and other foods. p53(+/-) mice (25/sex/group) received daily gavage exposure to vehicle only (negative control), resveratrol doses of 1000, 2000, or 4000 mg/kg/day, or p-cresidine (400 mg/kg/day; positive control). No mortality was seen in mice receiving the low dose of resveratrol. However, the mid and high doses induced mortality associated with impaction of the test article in the gastrointestinal tract. Resveratrol had no effect on body weight, food consumption, or clinical signs in surviving mice in any dose group, but induced dose-related increases in liver weight and serum cholesterol in both sexes. Mild anemia was seen in male mice at the high dose only; hematologic effects were not seen in females. Histopathology identified the kidney (hydronephrosis) and urinary bladder (epithelial hyperplasia) as target tissues for resveratrol toxicity. The incidences of both benign and malignant tumors in mice exposed to resveratrol were comparable to those in vehicle controls. By contrast, the positive control article, p-cresidine, induced urinary bladder cancer in both sexes. When administered to p53(+/-) mice at its maximum tolerated dose, resveratrol demonstrates no evidence of oncogenicity.     

A 28-day repeated dose toxicity study of ultraviolet absorber 2-(2'-hydroxy-3',5'-di-tert-butylphenyl) benzotriazole in rats

To examine the possible repeated-dose toxicity of an ultraviolet absorber, 2-(2'-hydroxy-3',5'-di-tert-butylphenyl)benzotriazole (HDBB), CD(SD)IGS rats were administered HDBB by gavage at a dose of 0 (vehicle: corn oil), 0.5, 2.5, 12.5, or 62.5 mg kg(-1) day(-1) for 28 days. At the completion of the administration period, a decrease in red blood cells, hemoglobin, and hematocrit was noted only in males at 2.5 mg/kg and more. Blood biochemical changes were noted at 0.5 mg/kg and more in males and at 62.5 mg/kg in females. Histopathologic changes were observed principally in the liver (vacuolar degeneration and hypertrophy of hepatocytes, bile duct proliferation, etc.) and in the heart (degeneration and hypertrophy of myocardium and cell infiltration). These changes were noted at 0.5 mg/kg and more in males and at 12.5 mg/kg and more in females. At higher doses, hypertrophy of tubular epithelium in the kidneys and diffuse follicular cell hyperplasia in the thyroids in both sexes and increased severity of basophilic tubules in the kidneys and extramedullary hematopoiesis in the spleen in males were also detected. After the 14-day recovery period, these changes mostly recovered in females but not in males. Based on these findings, no observed adverse effect level (NOAEL) was concluded to be less than 0.5 mg kg(-1) day(-1) in male rats and 2.5 mg kg(-1) day(-1) in female rats.     

Protective effects of Passiflora alata extract pretreatment on carbon tetrachloride induced oxidative damage in rats.

The leaf extract of Passiflora alata Dryander (P. alata) has been demonstrated to possess antioxidant activity in vitro. The aim of this study was to investigate the effects of P. alata leaf extract pretreatment on carbon tetrachloride-treated rats. Male Wistar rats were randomly allocated into four groups: group 1 (control - vehicle), group 2 and 3 (P. alata extract - 1 and 5mg/kg, respectively) and group 4 (trolox - 0.18mg/kg). Rats received daily pretreatment by oral gavage for 30 days followed by a single dose of CCl(4) (3ml/kg i.p. in vegetable oil) on the 30th day and were killed after 6h. The pretreatment with the P. alata extract provided significant protection to liver, evidenced by lower degree of necrosis, decreased lipid peroxidation (TBARS) and higher catalase and superoxide dismutase activities. Additionally, pretreated-rats with P. alata (5mg/kg) showed significantly decreased cardiac TBARS levels. Our results indicate that a low oral dose of P. alata leaf extract has both hepato and cardioprotective effects on rats treated with CCl(4).     

Effects of selected secondary metabolites of Fusarium moniliforme on unscheduled synthesis of DNA by rat primary hepatocytes.

The Fusarium moniliforme mycotoxins--fusarin C, fumonisin B1, moniliformin and bikaverin--were evaluated for genotoxicity by their ability to induce unscheduled DNA synthesis (UDS) in primary rat hepatocytes. Isolated hepatocytes were exposed to several concentrations of moniliformin (5.0-500 microM), bikaverin (1.0-500 microM), fumonisin B1 (0.5-250 microM), or fusarin C (1.0-100 microM). Aflatoxin B1, a known inducer of UDS, was included as a positive control. UDS was determined by autoradiography of cells after their exposure to [3H]thymidine. The highest doses of fusarin C and bikaverin caused cell death, but no cytotoxicity was observed in cells exposed to moniliformin or fumonisin B1. Fumonisin B1, moniliformin and bikaverin were not genotoxic in the UDS assay. The results of the UDS assay with fusarin C were inconclusive since a marginal effect on UDS was obtained.     

Mode of mutagenic action for the biocide Bioban CS-1246 in mouse lymphoma cells and implications for its in vivo mutagenic potential.

The biocidal agent, BIOBAN CS-1246 (7-ethyl bicyclooxazolidine, CAS# 7747-35-5, CS-1246) induced a concentration-dependent mutagenic response in mouse lymphoma (L5178Y TK+/-) cells both with and without the addition of S9 metabolic activation. Previous data indicating the ability of CS-1246 to hydrolyze in aqueous media to generate formaldehyde (FA), led us to investigate the potential role of FA in the CS-1246-induced mutagenic response in the mouse lymphoma assay (MLA). To accomplish this, the MLA on CS-1246 was repeated in the presence of a metabolizing system (formaldehyde dehydrogenase/NAD+), which was shown to successfully inhibit the mutagenic response of formaldehyde in this assay system. Significantly, the observed mutagenicity of CS-1246 was completely abrogated when the cultures were supplemented with formaldehyde dehydrogenase/NAD+, suggesting that the positive MLA response was attributable to the generation of FA in situ. These results demonstrate that in vitro mutagenicity of CS-1246 in the MLA is most likely associated with FA. Negative results from two in vivo assays for genotoxicity were consistent with the known activity of FA in these assays. In the mouse bone marrow micronucleus (MNT), there were no significant increases in micronucleated polychromatic erythrocytes (with evaluation of 2000/animal), after treatment with 0.5, 1, and 2 g/kg/day CS-1246 (6/dose group) for 2 consecutive days and sacrifice 24 h later. Furthermore, in the unscheduled DNA synthesis (UDS) study, male F344 rats (5 /dose group), given a single oral gavage (0, 1, and 2 g/kg) and evaluated at two time points (2-4 and 14-15 h post dosing), did not elicit an UDS response, indicating a lack of DNA reactivity in vivo. Based on the negative in vivo findings, it can be inferred that the FA detoxification mechanisms that exist in intact organisms prevent the likelihood of generating FA at levels capable of causing genotoxicity following exposure to CS-1246 at low, environmentally relevant concentrations. The extensive literature on FA would therefore be of value in assessing the carcinogenic risk to humans and animals from CS-1246 exposure.     

Effects of chronic valproate treatment on reproductive endocrine hormones in female and male Wistar rats

Valproate (VPA) has been claimed to induce endocrine disorders in both sexes in humans. There is sparse information regarding the mechanisms behind these disturbances. By using an animal model, we wanted to study the effect of valproate on hormonal function in non-epileptic rats. Female rats were given 0 (vehicle control, n=15), 200 mg/kg (n=15), or 300 mg/kg (n=20) valproate twice daily by gavage for 90 days, resulting in mean valproate concentrations within the therapeutic range 4–6 h after the last dose given. Serum testosterone concentrations remained unchanged, while estradiol levels were significantly reduced in both treatment groups, leading to significantly increased testosterone/estradiol ratios. Follicle stimulating hormone (FSH) levels remained unaltered in valproate treated rats, whereas the luteinizing hormone (LH) concentrations were reduced at the lowest valproate dose. Male rats received 0 (vehicle control, n=15), 200 mg/kg (n=15), or 400 mg/kg (n=20) valproate twice daily by gavage for 90 days, resulting in mean valproate concentrations within the therapeutic range 4–6 h after the last dose. Serum testosterone levels were not significantly changed, but there was a highly significant increase in FSH and LH concentrations at the high dose. In conclusion, the study demonstrates a drug-induced effect of valproate on endocrine function in both male and female rats. The results indicate that the drug exerts its effect primarily at the gonadal level, although a centrally mediated effect cannot be ruled out.