Protective effect of apigenin against N-nitrosodiethylamine (NDEA)-induced hepatotoxicity in albino rats.

A number of pharmacological properties have been attributed to apigenin. In the present study the effect of apigenin was investigated with respect to hepatotoxicity induced by N-nitrosodiethylamine (NDEA), a compound that is present in many food stuffs and has been reported to be a hepatocarcinogen. Male rats were exposed to NDEA (0.1mg/ml) dissolved in drinking-water separately, and with 10, 20, or 40mg/ml of apigenin for 21 days. The activity of glutamic-oxaloacetic transaminase (SGOT), glutamic-pyruvic transaminase (SGPT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) was measured in blood serum. Lipid peroxidation, protein carbonyl content and micronucleus frequency were determined in hepatocytes. To assess the effect on DNA damage, the comet assay was performed on hepatocytes, blood lymphocytes and bone-marrow cells of the exposed rats. The results of the study reveal that the treatment of NDEA together with apigenin showed a significant dose-dependent decrease in the serum concentration of the enzymes SGOT, SGPT, ALP and LDH (p<0.05). Histological sections of the liver also showed a protective effect of apigenin. A significant dose-dependent reduction in lipid peroxidation and protein carbonyl content was observed in rats exposed to NDEA (0.1mg/ml) together with apigenin (p<0.05). The results obtained for the comet assay in rat hepatocytes, blood lymphocytes and bone-marrow cells showed a significant dose-dependent decrease in the mean tail length (p<0.05). The present study supports the role of apigenin as an anti-genotoxic and hepatoprotective agent.

Garlic oil attenuated nitrosodiethylamine-induced hepatocarcinogenesis by modulating the metabolic activation and detoxification enzymes.

Nitrosodiethylamine (NDEA) is a potent carcinogen widely existing in the environment. Our previous study has demonstrated that garlic oil (GO) could prevent NDEA-induced hepatocarcinogenesis in rats, but the underlying mechanisms are not fully understood. It has been well documented that the metabolic activation may play important roles in NDEA-induced hepatocarcinogenesis. Therefore, we designed the current study to explore the potential mechanisms by investigating the changes of hepatic phase Ⅰ enzymes (including cytochrome P450 enzyme (CYP) 2E1, CYP1A2 and CYP1A1) and phase Ⅱ enzymes (including glutathione S transferases (GSTs) and UDP- Glucuronosyltransferases (UGTs)) by using enzymatic methods, real-time PCR, and western blotting analysis. We found that NDEA treatment resulted in significant decreases of the activities of CYP2E1, CYP1A2, GST alpha, GST mu, UGTs and increases of the activities of CYP1A1 and GST pi. Furthermore, the mRNA and protein levels of CYP2E1, CYP1A2, GST alpha, GST mu and UGT1A6 in the liver of NDEA-treated rats were significantly decreased compared with those of the control group rats, while the mRNA and protein levels of CYP1A1 and GST pi were dramatically increased. Interestingly, all these adverse effects induced by NDEA were simultaneously and significantly suppressed by GO co-treatment. These data suggest that the protective effects of GO against NDEA-induced hepatocarcinogenesis might be, at least partially, attributed to the modulation of phase I and phase II enzymes.

Effect of Chokeberry (Aronia melanocarpa) juice on the metabolic activation and detoxication of carcinogenic N-nitrosodiethylamine in rat liver.

Chokeberry is a rich source of polyphenols, which may counteract the action of chemical carcinogens. The aim of this study was to examine the effect of chokeberry juice alone or in combination with N-nitrosodiethylamine (NDEA) on phase I and phase II enzymes and DNA damage in rat liver. The forced feeding with chokeberry juice alone decreased the activities of enzymatic markers of cytochrome P450, CYP1A1 and 1A2. NDEA treatment also decreased the activity of CYP2E1 but enhanced the activity of CYP2B. Pretreatment with chokeberry juice further reduced the activity of these enzymes. Modulation of P450 enzyme activities was accompanied by the changes in the relevant proteins levels. Phase II enzymes were increased in all groups of animals tested. Chokeberry juice augmented DNA damage and aggravated the effect of NDEA. These results indicate that chokeberry may protect against liver damage; however, in combination with chemical carcinogens it might enhance their effect.

NDELA and nickel modulation of triazine disposition in skin.

Cutting fluids can become contaminated with metals (e.g., nickel, Ni) and nitrosamines (e.g., N-nitrosodiethanolamine, NDELA) and there is concern that these classes of contaminants can modulate dermal disposition and ultimately the toxicity of cutting fluid additives, such as irritant biocides (e.g., triazine). Biocides are added to these formulations to prevent bacterial degradation of commercial cutting fluids. The purpose of this study was to assess the dermal absorption and skin deposition of 14C-triazine when topically applied to porcine skin in an in vitro flow-through diffusion cell system as aqueous soluble oil (mineral oil, MO) or aqueous synthetic (polyethylene glycol, PEG) mixtures. 14C-Triazine mixtures were formulated with NDELA and/or Ni, or with a combination of three additional cutting fluid additives; namely, 5% linear alkylbenzene sulfonate (LAS), 5% triethanolamine (TEA) and 5% sulfurized ricinoleic acid. Neither Ni nor NDELA was absorbed during these 8-h studies. However, 14C-triazine absorption ranged from 2.72 to 3.29% dose in MO and 2.29-2.88% dose in PEG with significantly greater triazine absorption in MO than PEG when all additives and contaminates were present. The difference between these two diluents was most pronounced when NDELA and/or Ni were present in cutting fluids. These contaminants also enhanced triazine deposition on the skin surface and skin tissues especially with PEG-based mixtures. In essence, the dermal disposition of irritant biocides could be dependent on whether the worker is exposed to a soluble oil or synthetic fluid when these contaminants are present. Workers should therefore not only be concerned about dermatotoxicity of these contaminants, but also the modulated dermal disposition of cutting fluid additives when these contaminants are present in cutting fluid formulations.

Diversity of selective environmental substrates for human cytochrome P450 2A6: alkoxyethers, nicotine, coumarin, N-nitrosodiethylamine, and N-nitrosobenzylmethylamine.

Cytochrome P450 2A6 constitutes 5-10% of the total microsomal CYPs of human liver. Although CYP2A6 is the major coumarin 7-hydroxylase, other known substrates of CYP2A6 include many toxicants and precarcinogens. The chemical structure diversity of these substrates raises the question of their selectivity. Thus, kinetic parameters were determined for the hydroxylation of five substrates of diverse chemical structures known to be selective for cytochrome P450 2A6: methyl tert-butyl ether (MTBE), nicotine, coumarin, N-nitrosobenzylmethylamine (NBzMA), and N-nitrosodiethylamine (NDEA). Sources of enzymes were either human liver microsomes or heterologously expressed CYPs. Coumarin was shown to be the substrate with the highest affinity, followed by NDEA, nicotine, NBzMA, and MTBE. Variability of CYP2A6 catalytic activities in human liver was between 24-fold for MTBE to sevenfold for coumarin, while CYP2A6 content varied 68-fold in human liver microsomes. These five catalytic activities were highly significantly correlated between them and with hepatic CYP2A6 content. The most selective chemical inhibitor of these five substrates was shown to be 8-methoxypsoralen. Based upon chemical inhibition of the enzymatic activities of pure recombinant human CYPs, it cannot be totally excluded that P450s other than CYP2A6, especially CYP2E1, are involved, although to a lesser extent, in NDEA and NBzMA metabolism. In conclusion, the prototype probes for CYP2A6 phenotyping are coumarin and nicotine.

N-nitrosodiethylamine mechanistic data and risk assessment: bioactivation, DNA-adduct formation, mutagenicity, and tumor initiation.

N-Nitrosodiethylamine (NDEA) is DNA reactive after bioactivation and produces tumors in every animal species tested. Bioactivation is effected by several P450 isozymes including CYP2E1, which is ethanol inducible. Tumor formation in rat liver was proportional to O4-ethyldeoxythymidine formation in DNA, which was generally proportional to NDEA dose. At low doses in the 0.033-1.1 ppm range, the dose-response for esophageal tumor formation was sublinear, possibly due to DNA repair. Although no epidemiological studies have specifically evaluated NDEA, sufficient exposure levels would be expected to cause cancer in humans.

Short-term carcinogenesis bioassay of genotoxic procarcinogens in PIM transgenic mice.

E mu-pim-1 transgenic mice, which overexpress the pim-1 oncogene in lymphoid tissues, have shown increased susceptibility to induction of T cell lymphomas by N-ethyl-N-nitrosourea, a direct-acting chemical carcinogen (Nature, 340, 61-63, 1989). We sought to further evaluate E mu-pim-1 transgenic mice as a potential test animal for a short-term carcinogenesis bioassay. We chose to test four genotoxic procarcinogens; 2-acetylaminofluorene (2-AAF), N-nitro-sodiethylamine (NDEA), 1,2-dichloroethane (1,2-DCE) and benzene (BEN). These compounds require metabolic activation and, with the exception of benzene, are not mouse lymphomagens. Compounds were administered by gavage daily for 38 (NDEA and 2-AAF) or 40 (BEN and 1,2-DCE) weeks to groups of 25-29 male and female PIM mice at 1 and 3 mg/kg for NDEA, 50 and 100 mg/kg for BEN, 25-100 mg/kg for 2-AAF and 100-300 mg/kg for 1,2-DCE. Small but statistically significant increases in the incidence of malignant lymphoma were seen for three of the four carcinogens tested; in high dose males treated with 2-AAF, high and low dose females treated with NDEA and high dose females treated with 1,2-DCE. Results for BEN, the only mouse lymphomagen tested, did not show a statistically significant increase in the incidence of malignant lymphomas in transgenic mice within the 40 week duration of the study. NDEA also produced a high incidence (> 70%) of hepatic hemangiosarcomas in both sexes at the low and high dose levels. These results demonstrate that over-expression of the pim-1 oncogene in lymphoid tissue can confer susceptibility of this tissue to chemical carcinogenesis by genotoxic procarcinogens. However, whereas potent genotoxic carcinogens produced only small increases in the incidence of lymphoma and since BEN, a mouse lymphomagen, was negative, PIM transgenic mice may lack sufficient sensitivity to established carcinogens to justify their routine use in a short-term carcinogenesis screening assay.

Percutaneous penetration of N-nitrosodiethanolamine through human skin (in vitro): comparison of finite and infinite dose applications from cosmetic vehicles.

N-Nitroso compounds (nitrosamines) have been detected at the parts per billion level in a wide variety of matrices including industrial chemicals, pharmaceuticals, and food. Although N-nitrosodiethanolamine (NDELA) may be detected as an impurity in some cosmetic products, studies on NDELA absorption through human skin have been limited. A study to determine the extent of NDELA absorption following topical application was therefore undertaken to assist in the proper assessment of risk following unintended exposure. NDELA absorption was measured in vitro through human cadaver skin using isopropyl myristate (IPM) and generic prototype personal-care formulations (sunscreen and shampoo) spiked with [14C]NDELA. When applied as a finite dose at a concentration of 0.06% or lower, NDELA absorption was found to be a linear function of concentration. Total absorption at 48 hr ranged from approximately 35 to 65% of the dose and was formulation dependent (IPM > shampoo > or = sunscreen). Absorption occurred relatively rapidly from all formulations and peak rates of absorption were seen within the first 5 hr from the IPM and shampoo formulations. When applied as an infinite dose, total NDELA absorption followed a different rank order (shampoo > or = IPM > sunscreen) and evidence of barrier damage was seen with the shampoo formulation.

Clearance of N-nitrosodimethylamine and N-nitrosodiethylamine by the perfused rat liver. Relationship to the Km and Vmax for nitrosamine metabolism.

The first-pass clearance of dietary N-nitrosodimethylamine (NDMA) by the liver is the most important factor in the pharmacokinetics of this carcinogen in the rat, but is less important in the pharmacokinetics of N-nitrosodiethylamine (NDEA). The reason for the difference in clearance of these two nitrosamines is not known. These experiments were carried out to see whether the general characteristics of the clearance of these two carcinogens in vivo could be reproduced in the perfused liver, and whether the clearance could be correlated with the Michaelis-Menten parameters Km and Vmax for their metabolism. If this could be done one would be able to predict the possible extent of first-pass clearance of nitrosamines in man from measurement of Km and Vmax for nitrosamine metabolism by the human liver. The Km (22 microM) and Vmax (10.2 and 13.4 nmol/g liver/min) for the metabolism of NDMA by slices from two human livers, the inhibition of that metabolism by ethanol (Ki 0.5 microM), and the rate of N-7 methylation of DNA when slices are incubated with NDMA, were measured. These results are similar to those reported previously with rat liver. The Km (27 microM) for the metabolism of NDEA by rat liver slices and the inhibition of that metabolism by ethanol (Ki 1 microM) were estimated from the rate of ethylation of the DNA of the slices. The clearance of both these nitrosamines by the perfused rat liver was measured, and the results appeared to parallel those in vivo with a striking difference between the clearance of NDMA and NDEA. The maximal rate of clearance of NDMA was 11.2 nmol/g liver/min and of NDEA 8.9 nmol/g liver/min, similar to the Vmax for metabolism of NDMA by liver slices and to the estimated maximal rate of liver metabolism of both nitrosamines in the living rat. However, although the Km for metabolism of these two nitrosamines by liver slices is similar (about 25 microM), the logarithmic mean sinusoidal concentration [see Bass and Keiding, Biochem Pharmacol 37: 1425-1431, 1988] giving half maximal clearance during perfusion (the equivalent to Km) was 2.3 microM for NDMA and 10.6 microM for NDEA. The almost 5-fold difference between these two values is the basis for the difference between the clearance of the two nitrosamines.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes.

An acetyltransferase-overexpressing strain of Salmonella typhimurium (NM2009) has been used to investigate roles of human liver microsomal cytochrome P450 (P450) enzymes in the activation of carcinogenic nitrosamine derivatives, including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines, to genotoxic products. Studies employing correlation of activities with several P450-dependent monooxygenase reactions in different human liver samples, inhibition of microsomal activities by antibodies raised against human P450 enzymes and by specific P450 inhibitors, and reconstitution of activities with purified P450 enzymes suggest that the tobacco-smoke-related nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N-nitrosonornicotine (NNN) as well as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are oxidized to genotoxic products by different P450 enzymes, particularly P450 2E1 and 2A6. The activation of NDMA and NNN by liver microsomes was suggested to be catalyzed more actively by P450 2E1 than by other P450 enzymes because the activities were well correlated with NDMA N-demethylation and aniline p-hydroxylation in different human samples, and purified P450 2E1 had the highest activities in reconstituted monooxygenase systems. The relatively high contribution of P450 2A6 to the activation of NDEA and NNK was supported by the correlation seen with coumarin 7-hydroxylation in human liver microsomes, and antibodies raised against P450 2A6 inhibited both activities by approximately 50%. P450 3A4, 2D6 and 2C enzymes appear not to be extensively involved in the activation of these nitrosamines as judged by several criteria examined. Thus, this work indicates that several P450 enzymes, particularly P450 2E1 and 2A6, catalyze metabolic activation of nitrosamine derivatives including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines in human liver microsomes.

Participation of rat liver cytochrome P450 2E1 in the activation of N-nitrosodimethylamine and N-nitrosodiethylamine to products genotoxic in an acetyltransferase-overexpressing Salmonella typhimurium strain (NM2009).

The possible roles of cytochrome P450 (P450) enzymes in the metabolic activation of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) by rat liver microsomes have been examined in a system containing the bacterial tester strain Salmonella typhimurium NM2009, a newly developed strain showing high O-acetyltransfer activities. The DNA-damaging activity could be determined by measuring expression of the umu gene in a plasmid containing the fused umuC-lacZ gene construct in the bacteria. The following lines of evidence support the view that both NDMA and NDEA are principally oxidized to reactive products by P450 2E1 in rat liver microsomes. First, NDMA and NDEA were activated by rat liver microsomes in a protein- and substrate-dependent manner and the former chemical was more active than the latter; both activities were induced in rats treated with P450 2E1 inducers such as ethanol, acetone and isoniazid and by starvation. Second, activation of NDMA and NDEA were both inhibited significantly by antibodies raised against rat P450 2E1 and by P450 2E1 inhibitors such as diethyldithiocarbamate and 4-methylpyrazole in rat liver microsomes. Finally, in reconstituted monooxygenase systems containing purified rat P450 enzymes, P450 2E1 gave the highest rates of the activation of both NDMA and NDEA; the addition of rabbit cytochrome b5 to the system caused about a 1.5-fold increase in both reactions. In separate experiments we also found that N-nitrosomethylacethoxymethylamine, a compound that reacts with DNA after ester cleavage, is more genotoxic in S.typhimurium NM2009 than in S.typhimurium NM2000, a strain that is defective in O-acetyltransferase activity. Part of the pathway involved in the activation of nitrosamines is suggested to be acetylation of alkyldiazohydroxides formed by P450 or acetylesterase, because the genotoxic activity of N-nitrosomethylacethoxymethylamine in S.typhimurium NM2009 could be inhibited by the O-acetyltransferase inhibitor pentachlorophenol. These results indicate that NDMA and NDEA are oxidized to gentoxoic products by rat liver microsomes and that a P450 2E1 enzyme plays a major role in the activation of these two potent carcinogens. The activation pathway of N-nitrosodialkylamines through acetylation by O-acetyltransferase has been proposed. This simple bacterial system for measuring genotoxicity should facilitate studies on the activation of N-nitroso alkylamines.

The effect of caffeine on diethylnitrosamine-initiated hepatic altered foci in a mid-term induction system.

The modification potentials of caffeine on the development of preneoplastic hepatic enzyme altered foci were examined in an in vivo mid-term assay system. The number and area of glutathione S-transferase placental form-positive (GST-P+) hepatic foci was significantly reduced in rats given caffeine (0.1% or 0.2% in drinking water) followed by diethylnitrosamine (DEN) (200 mg/kg BW, IP) and DEN followed by caffeine as compared with the controls given carcinogen alone. Unscheduled DNA synthesis (USD) decreased approximately 70% in the hepatocytes treated with caffeine (200 mg/ml of medium). These results suggested that the antiinitiative effect of caffeine might be caused by the inhibition of the intracellular carcinogen accumulation and the antipromotive effect of caffeine might be associated with suppression of DNA repair.

[Modifying effect of antioxidants on the rate of lipid peroxidation in liver microsomes at early stages of diethylnitrosamine biotransformation]. / Modifitsiruiushchee vliianie antioksidantov na skorost' reaktsii perekisnogo okisleniia lipidov v mikrosomakh pecheni na nachal'nykh étapakh biotransformatsii diétilnitrozaminov.

Lipid peroxidation and free radicals' generation were investigated in rats treated with carcinogenic diethylnitrosamine (DENA) after pretreatment with some antioxidants. It was shown that decreased generation of free radicals during DENA metabolism in rat liver microsomes after pretreatment of animals with antioxidants may be the cause of the protective action of these antioxidants against DENA toxicity. Lipid peroxidation was shown to be not a crucial reaction in the toxic effect of DENA.

In-vivo activation of N-nitrosodiethanolamine and other n-nitroso-2-hydroxyalkylamines by alcohol dehydrogenase and sulfotransferase.

Alcohol dehydrogenase (ADH) activates N-nitrosodiethanolamine (NDELA) to a potent mutagen in the Ames mammalian microsome mutagenicity assay. In vivo, NDELA, its metabolite N-nitroso-2-hydroxymorpholine (NHMOR) and other 2-hydroxylated N-nitrosoalkylamines induce single-strand breaks in rat liver after a single oral application. After competitive inhibition of ADH by pretreatment with ethanol, induction of single-strand breaks by NDELA and N-nitroso(2-hydroxyethyl)ethylamine (NHEEA) was completely suppressed, whereas breaks induced by NHMOR were only partially reduced. Ethanol also influences cytochrome P450-dependent monooxygenases. To investigate whether the observed effect depends on inhibition of ADH and/or of monooxygenases, rats were pretreated with the ADH inhibitor 3-butylthiolane-1-oxide; a considerable reduction in the single-strand-break-inducing potential of NDELA was seen. Moreover, DNA damage induced by NDELA, NHMOR and other hydroxylated N-nitroso compounds is strongly reduced by pretreatment with the sulfotransferase inhibitor, 2,6-dichloro-4-nitrophenol (DCNP). DCNP pretreatment completely suppressed the induction of single-strand breaks by NDELA, whereas the number induced by NHEEA was only partially reduced. Our data suggest that ADH and sulfotransferase are enzymes responsible for the in-vivo activation of N-nitroso-2-hydroxyalkylamines.

Metabolism and cellular interactions of N-nitrosodiethanolamine.

N-Nitrosodiethanolamine (NDELA) labelled with 14C at the alpha carbon was administered by gavage to adult male Fischer 344 rats at various doses ranging from 0.6 to 100 mg per rat. The proportion of the dose excreted as 14CO2 was small, ranging from 0.27% at the lowest dose to 0.83% at the highest in 24 h. At all doses, approximately 95% of the dose of radioactivity (most of which was NDELA) appeared in the urine within 24 h, but the proportion of metabolites increased from 7% to 14% from the lowest to the highest dose. The specific activity of the nucleic acids isolated from the liver of rats given 100 mg and 100 microCi of NDELA was very low and was the same at 6 h and 24 h after treatment (70 dpm/mg DNA, 92-95 dpm/mg RNA). N7-(2-Hydroxyethyl)guanine and O6-(2-hydroxyethyl)-guanine were tentatively identified in the hydrolysates of the nucleic acids, comprising 10% and 4%, respectively, of the DNA radioactivity; there was no difference between the amounts found 6 h and 24 h after NDELA treatment. In addition to NDELA, four components were separated from rat urine, and two were identified. One is the glucuronide of NDELA, the other is N-nitroso-N-(2-hydroxyethyl)carboxymethylamine. Neither nitroso-2-hydroxymorpholine nor a sulfate of NDELA was detected.