[Health risk assessment associated with priority potentially hazardous chemical compounds detected in canned meat and meat-and-vegetable food for infants].

Assessment of public health safety associated with chemical contaminants consumed with food is an important component for solving the tasks of ensuring the sanitary and epidemiological well-being of the population. For these purposes, it is necessary to establish priority potentially dangerous compounds among the identified undeclared and unintended chemical contaminants for further consumers risk assessment. In conditions of unacceptable levels of health risk, it is necessary to decide whether it is advisable to develop new or change existing hygiene standards for these substances. The aim of the study was to assess the health risk associated with priority potentially dangerous unintended chemical components of contamination in canned meat and meat-and-vegetable products for infants (using the example of N-nitrosoamines). Material and methods. The selection of priority chemical compounds and public health risk assessment were carried out in accordance with the procedure for identifying undeclared and potentially dangerous unintended chemicals in food and using modified approaches based on the results of previous studies. Health risk assessment for infants consuming canned meat and meat-and-vegetables containing priority chemicals was carried out in accordance with the methodology approved by the Eurasian Economic Commitee, as well as using the Guidelines for assessing risks to public health when exposed to environmental pollutants. To characterize the calculated risk levels, the classification proposed in the draft document «Guidelines for assessing risks to public health when exposed to environmental pollutants¼ was used. Results. N-nitrosoamines are classified as chemical contaminants that are a priority for public health risk assessment, identified on the basis of the potential hazard category. A comparative health risk assessment for the target group consumers of canned meat and meat-and-vegetables sold on the territory of the Russian Federation and the Socialistic Republic of Vietnam showed the presence of an unacceptable risk to the health of the Russian population in relation to non-carcinogenic adverse health effects associated with the intake of N-nitrosodimethylamine (NDMA) (hazard quotient HQ=1.1) and N-nitrosodibuthylamine (HQ=5.25). Many experimental (animal) researches confirm the formation of negative effects and potential harm to humans, formed by the consumption of N-nitrosoamines with food. At the same time, the level of the exposure of N-nitrosoamines during oral administration was assessed mainly from the side of carcinogenic effects, however, this study reflects the hazard, including from non-carcinogenic risks associated not only with NDMA and N-nitrosodiethylamine, for which there is a hygienic standard, but also for other N-nitrosoamines, for which there is no hygienic regulation. In turn, the European Food Safety Agency's (EFSA) study notes that it is necessary to conduct a risk assessment for human health in relation to other N-nitrosoamines identified in food, and, if necessary, rationing of these compounds, which is also confirmed by this study. Conclusion. The conducted health risk assessment of the target population of Russia (infants from 6 months to 3 years) consuming the studied types of products containing N-nitrosoamines showed the presence of unacceptable levels of non-carcinogenic health risks in relation to the development processes and impaired liver function. In connection with the identified risks to the health of the target population, it is advisable to establish maximum permissible levels not only for the amount of NDMA and N-nitrosodiethylamine, but also for other N-nitrozoamines identified by the results of the study, with their subsequent regulation.

Standardization of diethylnitrosamine-induced hepatocellular carcinoma rat model with time based molecular assessment.

This study was intended (1) to develop a robust animal model for hepatocellular carcinoma (HCC) research, in which HCC tumors develop in a background of fibrosis or cirrhosis; and (2) to explore time-dependent regulatory changes in key molecular markers during disease advancement and HCC development. With the aim of establishing such HCC model, male Sprague-Dawley rats were injected with diethylnitrosamine (DEN) at a dose of 30 mg/kg twice a week for 10 weeks then once a week from 12th to 16th weeks. The rats were kept under observation until 18th week. At defined time intervals (2nd, 4th, 12th, and 18th week), serum biomarkers and microscopic components of tissue samples were used to investigate the chronic progression of liver disease, while gene and protein analysis was used to monitor expression patterns during HCC development. DEN-intoxicated rats manifested inflammation at week 4, fibrosis at week 12 and cirrhosis with early HCC tumors at week 18. Molecular analysis revealed that key markers of inflammation (Il-1ß, Il-6, and Tnf-α), fibrosis (Tgf-ß1, Col1α1, Col3α1, and Timp-1), and angiogenesis (Hif1-α and Vegf) were promptly (P ≤ 0.001) up-regulated at week 4, week 12 and week 18, respectively. Oxidative stress (iNos, Cyp2e1, and Sod1) and pro-apoptotic (Bax) markers showed significant upregulation from week 4 to week 12. However, Sod1 and Bax expressions dropped after week 12 and reached a minimum at 18th week. Strikingly, expressions of anti-apoptotic (Bcl-2) and cell proliferation (Pcna, Hgf, and Afp) markers were abruptly increased at week 18. Collectively, we describe an 18-week HCC model in DEN-intoxicated rats that exhibit chronic inflammation, oxidative imbalance, advance fibrosis/cirrhosis, halted apoptosis, and angiogenic sprouting, progressively.

Assessment of SENP3-interacting proteins in hepatocytes treated with diethylnitrosamine by BioID assay.

SUMOylation of proteins regulates cell behaviors and is reversibly removed by small ubiquitin-like modifier (SUMO)-specific proteases (SENPs). The SENP family member SENP3 is involved in SUMO2/3 deconjugation and has been reported to sense cell stress and accumulate in several human cancer cells and macrophages. We previously reported that Senp3-knockout heterozygous mice showed smaller liver, but the pertinent mechanisms of SENP3 and SUMOylated substrates remain unclear. Thus, in this study, we investigated the interacting proteins with SENP3 and the alteration in hepatocytes treated with the xenobiotic diethylnitrosamine (DEN), which is specifically transformed in the liver and induces DNA double-strand breaks. Our data revealed that a certain amount of SENP3 was present in normal, untreated hepatocytes; however, DEN treatment promoted rapid SENP3 accumulation. SENP3 was mainly localized in the nuclei, and its level was significantly increased in the cytoplasm after 2 h of DEN treatment. The application of the recent proximity-dependent biotinylation (BioID) method led to the identification of 310 SENP3-interacting proteins that were involved in not only gene transcription but also RNA splicing, protein folding, and metabolism. Furthermore, after DEN exposure for a short duration, ribosomal proteins as well as proteins associated with mitochondrial ATP synthesis, membrane transport, and bile acid synthesis, rather than DNA repair proteins, were identified. This study provides insights into the diverse regulatory roles of SENP3, and the BioID method seems to be efficient for identifying physiologically relevant insoluble proteins.

B-mode ultrasound for the assessment of hepatic fibrosis: a quantitative multiparametric analysis for a radiomics approach.

Hepatic fibrosis and cirrhosis are a growing global health problem with increasing mortality rates. Early diagnosis and staging of hepatic fibrosis represent a major challenge. Currently liver biopsy is the gold standard for fibrosis assessment; however, biopsy requires an invasive procedure and is prone to sampling error and reader variability. In the current study we investigate using quantitative analysis of computer-extracted features of B-mode ultrasound as a non-invasive tool to characterize hepatic fibrosis. Twenty-two rats were administered diethylnitrosamine (DEN) orally for 12 weeks to induce hepatic fibrosis. Four control rats did not receive DEN. B-mode ultrasound scans sampling throughout the liver were acquired at baseline, 10, and 13 weeks. Computer extracted quantitative parameters representing brightness (echointensity, hepatorenal index) and variance (heterogeneity, anisotropy) of the liver were studied. DEN rats showed an increase in echointensity from 37.1 ± SD 7.8 to 53.5 ± 5.7 (10 w) to 57.5 ± 6.1 (13 w), while the control group remained unchanged at an average of 34.5 ± 4.5. The three other features studied increased similarly over time in the DEN group. Histologic analysis showed METAVIR fibrosis grades of F2-F4 in DEN rats and F0-F1 in controls. Increasing imaging parameters correlated with increasing METAVIR grades, and anisotropy showed the strongest correlation (ρ = 0.58). Sonographic parameters combined using multiparametric logistic regression were able to differentiate between clinically significant and insignificant fibrosis. Quantitative B-mode ultrasound imaging can be implemented in clinical settings as an accurate non-invasive tool for fibrosis assessment.

Lesson Learnt from Recall of Valsartan and Other Angiotensin II Receptor Blocker Drugs Containing NDMA and NDEA Impurities.

The presence of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) impurities in angiotensin II receptor blocker (ARB) drugs containing tetrazole ring has triggered worldwide product recalls. The purpose of this article is to identify the potential gap area in current pharmaceutical industry practice that might have led to the NMDA and NDEA impurities escaping the drug manufacturer's and FDA's attention. The impact of process change was not adequately assessed by the manufacturer of contaminated APIs (active pharmaceutical ingredients), and potential for generation of mutagenic or other toxic impurities was not considered. The safety and risk associated with a chemical synthetic process was also not evaluated. This is primarily due to current industry practice which focuses on controlling the impurities above reporting threshold. ICH Q3A and FDA guidance on genotoxic and carcinogenic impurities in drug substances and products need to be integrated so that the ICH Q3A decision tree (attachment 3) begins by checking whether the synthetic process has been evaluated for the potential to generate toxic impurities. The compliance with ICH Q3A limits should be carried out only after the process has been determined to be safe without the risk of generating mutagenic and carcinogenic impurities.

Risk assessment of N-nitrosodiethylamine (NDEA) and N-nitrosodiethanolamine (NDELA) in cosmetics.

N-nitrosamines and their precursors found in cosmetics may be carcinogenic in humans. Thus the aim of this study was to carry out risk assessment for N-nitrosamines (N-nitrosodiethanolamine [NDELA], N-nitrosodiethylamine [NDEA]) and amines (triethanolamine [TEA], diethanolamine [DEA]) levels in cosmetics determined using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedures. NDELA and NDEA concentrations were present at levels of "not detected" (N.D.) to 596.5 µg/kg and N.D. to 40.9 µg/kg, respectively. TEA and DEA concentrations ranged from N.D. to 860 µg/kg and N.D. to 26.22 µg/kg, respectively. The nitrite concentration (3-2250 mg/l), number of nitrosating agents to a maximum 5, and pH (3.93-10.09) were also assessed. The impact of N-nitrosamine formation on the levels of TEA, DEA, nitrite, and other nitrosating agents was also examined. N-nitrosamine concentrations correlated with the number of nitrosating agents and nitrite concentrations. Data demonstrated that higher nitrite concentrations and a greater number of nitrosating agents increased NDELA and NDEA yields. Further, the presence of TEA and DEA exerted a significant influence on N-nitrosamine formation. Risk assessments, including the margin of exposure (MOE) and lifetime cancer risk (LCR) for N-nitrosamines and margin of safety (MOS) for amines, were calculated using product type, use pattern, and concentrations. Exposure to maximum amounts of NDELA and NDEA resulted in MOE > 10,000 (based upon the benchmark dose lower confidence limit 10%) and LCR <1 × 10-5, respectively. In addition, TEA and DEA concentrations in cosmetic samples resulted in MOS values >100. Therefore, no apparent safety concerns were associated with cosmetic products containing NDELA, NDEA, TEA, and DEA in this study. However, since amines and nitrosating agents produce carcinogenic nitrosamines, their use in cosmetics needs to be minimized to levels as low as technically feasible.

Activation of SRY accounts for male-specific hepatocarcinogenesis: Implication in gender disparity of hepatocellular carcinoma.

Sex affects the risk, treatment responses and outcome of many types of cancers. The mechanism of gender disparity in development of hepatocellular carcinoma (HCC) remains obscure. Sex-determining region on Y chromosome (SRY) was overexpressed in approximate 84% male patient HCC. Moreover, we are the first to generate a liver-specific transgenic (TG) murine model with overexpression of the male specific gene SRY. Subject to a single intraperitoneal injection N-nitrosodiethylamine (DEN) at day 14, TG and wildtype (WT) mice of both genders were sacrificed at different time points (6-13.5 months). Overexpression of SRY in male TG and ectopic expression of SRY in female TG livers promoted DEN-induced hepatocarcinogenesis compared to age- and sex-matched WT. This accelerated tumorigenesis in TG of both genders was a consequence of increased injury and inflammation, fibrosis, and compensatory enhancement in hepatocytes proliferation secondary to activation of downstream targets Sox9 and platelet-derived growth factor receptor α (PDGFRα)/phosphoinositide 3-kinase (PI3K)/Akt and c-myc/CyclinD1. In conclusion, activation of SRY and its downstream Sox9 and PDGFRα pathways are commonly involved in male hepatocarcinogenesis, which provides novel insights into gender disparity and sex-specific therapeutic strategies of HCC.

Effect of Pomegranate Hull Extract on Liver Neoplastic Changes in Rats: More than an Antioxidant.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide. The current work was designed to elucidate the molecular mechanisms underlying the antitumorigenic effect of pomegranate hull extract (PHE) in livers of rats exposed to the hepatocarcinogen diethyl nitrosamine (DENA) with emphasis on oxidative stress, proliferation, and apoptosis. Male albino rats were divided into three groups: normal control, DENA group, and PHE group. PHE was given to rats orally 3 times weekly for 10 wk, 4 wk before and 6 wk after DENA (200 mg/kg, single i.p. dose). The results indicated a prophylactic effect of PHE against neoplastic changes in the liver, which was evidenced by the decrease of tumor size, liver index, and the anti-apoptotic protein Bcl-2; and the increase of glutathione. PHE group also showed decreased expression of liver cyclin D1 and ß-catenin genes compared with DENA group. It is proved that PHE has antitumorigenic effect and could be a candidate for anticancer drugs.

Qualitative and quantitative approaches in the dose-response assessment of genotoxic carcinogens.

Qualitative and quantitative approaches are important issues in field of carcinogenic risk assessment of the genotoxic carcinogens. Herein, we provide quantitative data on low-dose hepatocarcinogenicity studies for three genotoxic hepatocarcinogens: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and N-nitrosodiethylamine (DEN). Hepatocarcinogenicity was examined by quantitative analysis of glutathione S-transferase placental form (GST-P) positive foci, which are the preneoplastic lesions in rat hepatocarcinogenesis and the endpoint carcinogenic marker in the rat liver medium-term carcinogenicity bioassay. We also examined DNA damage and gene mutations which occurred through the initiation stage of carcinogenesis. For the establishment of points of departure (PoD) from which the cancer-related risk can be estimated, we analyzed the above events by quantitative no-observed-effect level and benchmark dose approaches. MeIQx at low doses induced formation of DNA-MeIQx adducts; somewhat higher doses caused elevation of 8-hydroxy-2'-deoxyquanosine levels; at still higher doses gene mutations occurred; and the highest dose induced formation of GST-P positive foci. These data indicate that early genotoxic events in the pathway to carcinogenesis showed the expected trend of lower PoDs for earlier events in the carcinogenic process. Similarly, only the highest dose of IQ caused an increase in the number of GST-P positive foci in the liver, while IQ-DNA adduct formation was observed with low doses. Moreover, treatment with DEN at low doses had no effect on development of GST-P positive foci in the liver. These data on PoDs for the markers contribute to understand whether genotoxic carcinogens have a threshold for their carcinogenicity. The most appropriate approach to use in low dose-response assessment must be approved on the basis of scientific judgment.

Assessment of a twice dosing regimen both before and after partial hepatectomy in the rat liver micronucleus test.

The liver micronucleus test is an important method to detect in vivo genotoxicants, especially those that require metabolic activation for their genotoxicity. We have already reported that structural or numerical chromosome aberration inducers have to be given before or after partial hepatectomy, respectively, to detect their genotoxicity in the liver of rats. In the present study, we assessed a twice dosing regimen, in which the genotoxicant is dosed both before and after partial hepatectomy, using the four chromosome aberration inducers used in the previous study. Two structural chromosome aberration inducers (diethylnitrosamine and 1,2-dimethylhydrazine) and two numerical chromosome aberration inducers (colchicine and carbendazim) were used. The genotoxicant was administered to 8-week old male F344 rats one day before and again one day after the partial hepatectomy and hepatocytes were isolated 3 days after second dosing (4 days after the partial hepatectomy). As a result, all genotoxicants (structural or numerical chromosome aberration inducers) caused a dose-dependent statistically significant increase in the incidence of micronucleated hepatocytes when given both before and after partial hepatectomy. No marked difference was observed in general toxicity, relative liver weight and cell classification between single dosing regimens and twice dosing regimen of the genotoxicants. These results confirm that the twice dosing regimen, in which the test compound is dosed both before and after partial hepatectomy, can detect in vivo induction of micronucleated hepatocytes by structural or numerical chromosome aberration inducers qualitatively similar to their appropriate regimen in which the test compound is administered either before or after partial hepatectomy.

Assessment of angiogenesis of hepatocellular carcinoma using dynamic contrast enhanced MR and histopathologic correlation in an experimental rat model.

BACKGROUND/AIMS: To assess the perfusion parameters and angiogenesis of HCC using dynamic contrast enhanced(DCE) MR and to correlate it with histopathologic findings in an experimental rat model. METHODOLOGY: Twenty rats were continuously infused with diethylnitrosamine (DEN) for tumor induction. After 32 to 36 weeks of DEN treatment, the rats underwent MRI of the liver with a 3-T MR imaging system. Perfusion parametric maps and perfusion parameters such as, time to peak (TTP) and peak enhancement (PE) were obtained by using a commercially available software package. The nodules were correlated precisely to DCE MR images. RESULTS: A total of 13 nodules were found in 12 rats; 5 dysplastic nodule (DN)s were identified in 5 rats and 8 HCCs (3 Edmonson grade I, 2 Edmonson grade I-II, 3 Edmonson grade II) were found in 7 rats. There were significant differences in mean values of PE and HPH (histogram peak height) of PE between DN and HCC. Mean value and HPH of PE showed statistically significant correlation with tumor grade. CONCLUSIONS: There were significant differences in perfusion parameters between DN and HCC. DCE MR imaging can be used in the differential diagnosis and management of liver disease in hepatocarcinogenesis.

Assessment of genotoxicity induced by 7,12-dimethylbenz(a)anthracene or diethylnitrosamine in the Pig-a, micronucleus and Comet assays integrated into 28-day repeat dose studies.

As part of the Stage 3 of the Pig-a international trial, we evaluated 7,12-dimethylbenz(a)anthracene (DMBA) for induction of Pig-a gene mutation using a 28-day repeat dose study design in Sprague-Dawley rats. In the same study, chromosomal damage in peripheral blood and primary DNA damage in liver were also investigated by the micronucleus (MN) assay and the Comet assay, respectively. In agreement with previously published data (Dertinger et al., [2010]: Toxicol Sci 115:401-411), DMBA induced dose-dependent increases of CD59-negative erythrocytes/reticulocytes and micronucleated reticulocytes (MN-RETs). However, there was no significant increase in DNA damage in the liver cells when tested up to 10 mg/kg/day, which appears to be below the maximum tolerated dose. When tested up to 200 mg/kg/day in a follow-up 3 dose study, DMBA was positive in the liver Comet assay. Additionally, we evaluated diethylnitrosamine (DEN), a known mutagen/hepatocarcinogen, for induction of Pig-a mutation, MN and DNA damage in a 28-day study. DEN produced negative results in both the Pig-a mutation assay and the MN assay, but induced dose-dependent increases of DNA damage in the liver and blood Comet assay. In summary, our results demonstrated that the Pig-a mutation assay can be effectively integrated into repeat dose studies and the data are highly reproducible between different laboratories. Also, integration of multiple genotoxicity endpoints into the same study not only provides a comprehensive evaluation of the genotoxic potential of test chemicals, but also reduces the number of animals needed for testing, especially when more than one in vivo genotoxicity tests are required.

Carcinogenic risk assessment: are there dose thresholds for carcinogens?

While it has been generally accepted that genotoxic carcinogens have no dose threshold for their carcinogenic potential, there is increasing evidence that very low doses in fact are incapable of inducing tumours or preneoplastic lesions. Thus not only so-called epigenetic 'non-genotoxic' compounds like phenobarbital and benzene hexachloride, but also unequivocally genotoxic carcinogens like the heterocyclic amines, 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline and amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and the nitrosamines diethylnitrosamine, and dimethylnitrosamine, may exhibit a practical dose threshold below which they do not induce histopathologically assessable lesions. Some form of physiological adaptation may thus be expected to occur in response to low doses of all types of DNA-damaging agents. With 'non-genotoxic' agents there may even be hormesis or paradoxical protection at very low dose.

Assessment of the effect of betaine on p16 and c-myc DNA methylation and mRNA expression in a chemical induced rat liver cancer model.

BACKGROUND: The development and progression of liver cancer may involve abnormal changes in DNA methylation, which lead to the activation of certain proto-oncogenes, such as c-myc, as well as the inactivation of certain tumor suppressors, such as p16. Betaine, as an active methyl-donor, maintains normal DNA methylation patterns. However, there are few investigations on the protective effect of betaine in hepatocarcinogenesis. METHODS: Four groups of rats were given diethylinitrosamine (DEN) and fed with AIN-93G diets supplemented with 0, 10, 20 or 40 g betaine/kg (model, 1%, 2%, and 4% betaine, respectively), while the control group, received no DEN, fed with AIN-93G diet. Eight or 15 weeks later, the expression of p16 and c-myc mRNA was examined by Real-time PCR (Q-PCR). The DNA methylation status within the p16 and c-myc promoter was analyzed using methylation-specific PCR. RESULTS: Compared with the model group, numbers and areas of glutathione S-transferase placental form (GST-p)-positive foci were decreased in the livers of the rats treated with betaine (P < 0.05). Although the frequency of p16 promoter methylation in livers of the four DEN-fed groups appeared to increase, there is no difference among these groups after 8 or 15 weeks (P > 0.05). Betaine supplementation attenuated the down-regulation of p16 and inhibited the up-regulation of c-myc induced by DEN in a dose-dependent manner (P < 0.01). Meanwhile, increases in levels of malondialdehyde (MDA) and glutathione S-transferase (GST) in model, 2% and 4% betaine groups were observed (P < 0.05). Finally, enhanced antioxidative capacity (T-AOC) was observed in both the 2% and 4% betaine groups. CONCLUSION: Our data suggest that betaine attenuates DEN-induced damage in rat liver and reverses DEN-induced changes in mRNA levels.

O papel das enzimas CYP2A na susceptibilidade ao câncer do esôfago e sua regulação / The role of CYP2A enzymes in susceptibility to esophageal cancer and its regulation

As nitrosaminas são pré-carcinógenos presentes no cigarro e em alguns alimentos e são os únicos compostos capazes de induzir tumores no esôfago de animais experimentais. Uma vez que as nitrosaminas somente se tornam cancerígenos após serem metabolizadas por enzimas CYP, a presença de um CYP capaz de ativá-las é fundamental para a susceptibilidade tecidual à indução de tumores por estes carcinógenos. A indução de tumores esofágicos pelas nitrosaminas varia entre os animais experimentais, sendo o rato a espécie mais susceptível. Em 2001, nosso grupo identificou o CYP2A3 como sendo a principal enzima responsável pela ativação da NDEA no esôfago de ratos. Por outro lado, nenhuma das nitrosaminas testadas até o momento induz tumores esofágicos no hamster, sendo o fígado o principal órgão-alvo. Com o objetivo de achar uma explicação mecanística para a resistência do esôfago do hamster às nitrosaminas, neste trabalho nós caracterizamos a expressão de enzima CYP2A no esôfago deste animal e comparamos o metabolismo da NDEA entre esôfago e fígado. Mostramos que tanto o esôfago quanto o fígado expressam os mRNAs dos CYP2A8, CYP2A9 e CYP2A16. A expressão protéica, no entanto, é diferente entre os tecidos. Os resultados de Western blotting mostram que, apesar do esôfago e fígado expressarem uma isoforma em comum, o esôfago expressa uma segunda isoforma que não é presente no fígado, enquanto o fígado expressa uma proteína que não é detectada no esôfago. Surpreendentemente, também detectamos uma alta ativação da NDEA pelos microssomos esofágicos. Porém, a eficiência catalítica desta reação foi cerca de 40 vezes menor do que a detectada nos microssomos hepáticos. Um anticorpo anti-CYP2A6 humano foi capaz de inibir o metabolismo da NDEA em microssomos esofágicos, mas não hepáticos. A diferença na eficiência catalítica da reação de NDEA entre esôfago e fígado pode explicar porque as nitrosaminas nunca induzem tumores esofágicos em hamsters. Devido ao papel dos CYP2A...

Nitrosamines are pre-carcinogens found in food and cigarette smoke and are the only compounds known to induce esophageal tumors in experimental animals. Nitrosamines become active carcinogens tumors only after being metabolized by CYP enzymes. Therefore, CYP expression is essential for tissue-specific tumor induction by nitrosamines. Esophageal tumor induction by nitrosamines varies amongst experimental animals, with the rat being the most sensitive species. We have previously shown that CYP2A3 is expressed in the rat esophagus and that CYP2A3 is responsible for NDEA activation in this tissue. On the other hand, none of the nitrosamines tested so far induces esophageal tumors in hamsters, with the liver being the main target-organ for nitrosamine induced tumors. In order to find a mechanistic explanation for its esophageal resistance to nitrosamines, we have characterized CYP2A expression in hamster esophagus and liver and compared NDEA metabolism between these tissues. Hamster esophagus and liver express CYP2A8, CYP2A9 and CYP2A16 mRNAs. However, protein expression is different between the tissues, and our Western blotting results showed that, whereas both the esophagus and liver express two CYP2A isoforms each, only one of the isoforms is similar in both tissues. Surprisingly, we have detected a high NDEA activation in the esophageal microsomes. However, the catalytic efficiency for this reaction was about 40-fold lower than the one detected for hepatic microsomes. An antibody against human CYP26 was able to inhibit NDEA in hamster esophageal, but not liver microsomes. The difference in the catalytic efficiency towards NDEA metabolism between esophagus and liver could help to explain hamster esophageal resistance to nitrosamines. CYP2A inhibition could be a promising approach in chemoprevention, leading to a reduction of the diseases associated with tobacco smoking. There are few data about CYP2A inhibition in experimental animals...

Bioassay of mannitol and caprolactam and assessment of response to diethylnitrosamine in heterozygous p53-deficient (+/-) and wild type (+/+) mice.

Alternative bioassays of mannitol (MAN) and caprolactam (CAP) were conducted in transgenic p53-deficient mice. Also, to assess the sensitivity of the transgenic mice to a model DNA-reactive carcinogen, the hepatic effects of diethylnitrosamine (DEN) were compared in the wild type background strain of mouse and in the transgenic derivative. Fifty-one male wild type strain C57BL/6 mice p53 (+/+), 8 weeks old, and 51 heterozygous p53 (+/-) C57BL/6 Tac-[KO] Trp53 N5 mice, 8 weeks old, were allocated to six experimental groups as follows: groups 1 (wild type +/+) and 2 (p53 +/-) served as room controls, groups 3 (+/+) and 4 (+/-) were exposed orally (gavage) to 50 mumol/kg body weight DEN weekly for a total of ten doses during the first 10 weeks of the study, group 5 (+/-) was exposed to 15,000 ppm CAP in the diet for up to 26 weeks, and group 6 (+/-) was exposed to 50,000 ppm MAN in the diet for up to 26 weeks. After 10 weeks, liver from control and DEN-exposed mice was used for O4-ethylthymidine (O4-EtT) DNA adduct analysis by the immunoslot blot method. The cell replicating fraction (RF) in the liver was determined by quantification of the percentage of immunohistochemically stained hepatocytes positive for proliferating cell nuclear antigen. No significant or consistent body or liver weight changes were present in any of the treatment groups. No consistent and pertinent changes in RF values were present in any of the treatment groups. None of the tested substances produced neoplasms of any type in p53 (+/-) mice. DEN induced comparable levels of O4-EtT adducts in the liver in both wild type and p53 +/- genotypes, but no morphologic changes were evident in the livers of either genotype. The lack of response to DEN, in spite of formation of DNA adducts, may reflect the resistance to hepatocarcinogenesis of the background C57BL/6 strain of the transgenic, and calls into question the general sensitivity of this transgenic for detection of carcinogenic effects.

Induction of esophageal carcinogenesis by diethylnitrosamine and assessment of the promoting effect of ethanol and N-nitrosonornicotine: experimental model in mice.

It is known that some nitrosamines preferably affect particular organs because of their organospecificity. Diethylnitrosamine (DEN) is one of the most powerful nitrosamines for experimentally inducing esophagus cancer. The present study aimed to evaluate the rate and type of epithelial lesions induced by DEN in mice. We also assessed the role of alcohol and N-nitrosonornicotine (NNN) as promoters of this carcinogenesis. A total of 208 female mice (Mus musculus) were allocated to five experimental groups: group 1, water only (controls); group 2, DEN + water; group 3, DEN + NNN; group 4, DEN + 6% alcohol solution; group 5, DEN + NNN + 6% alcohol solution. Animals in groups 2, 3, 4 and 5 received DEN (0.04 ml/l) three times per week, and during the following 4 days they received the other solutions. NNN was provided at a final concentration of 30 mg/l. The overall experimental period was 180 days. At the end of this time, the animals were killed and their esophagus was dissected for macro- and microscopic analysis. There was no significant difference in relation to the size of the esophagus and to the average DEN intake by the animals (p > 0.05). A statistically significant difference (p < 0.0001) was observed between controls and all other experimental groups. There was no significant difference among experimental groups treated with carcinogens (p > 0.05). The average incidence of cancer was 85.4%. The experimental model used in the present study is a very potent indicator of esophagus cancer. Owing to the high incidence for cancer observed in the present study, it was not possible to assess the effect of alcohol and NNN as inducers for the development of esophageal cancer.

Environmental complex mixture toxicity assessment.

Trichloroethylene (TCE) was found as a contaminant in the well supplying water to an aquatic testing laboratory. The groundwater was routinely screened by a commercial laboratory for volatile and semivolatile compounds, metals, herbicides, pesticides, and polychlorinated biphenyls using U.S. Environmental Protection Agency methods. Although TCE was the only reportable peak on the gas chromatograph, with average concentrations of 0.200 mg/l, other small peaks were also present, indicating the possibility that the contamination was not limited to TCE alone. A chronic 6-month carcinogenicity assay was conducted on-site in a biomonitoring trailer, using the Japanese medaka fish (Oryzias latipes) in an initiation-promotion protocol, with diethylnitrosamine (DEN) as the initiator and the TCE-contaminated groundwater as a promoter. Study results indicated no evidence of carcinogenic potential of the groundwater without initiation. There was, however, a tumor-promotional effect of the groundwater after DEN initiation. A follow-up laboratory study was conducted using reagent grade TCE added to carbon-filtered groundwater to simulate TCE concentrations comparable to those found in the contaminated groundwater. Study results indicated no promotional effects of TCE. These studies emphasize the necessity for on-site bioassays to assess potential environmental hazards. In this instance, chemical analysis of the groundwater identified TCE as the only reportable contaminant, but other compounds present below reportable limits were noted and may have had a synergistic effect on tumor promotion observed with the groundwater exposure. Laboratory toxicity testing of single compounds can produce toxicity data specific to that compound for that species but cannot take into account the possible toxic effects of mixtures of compounds.

Revalidation of the in vitro alkaline elution/rat hepatocyte assay for DNA damage: improved criteria for assessment of cytotoxicity and genotoxicity and results for 81 compounds.

The in vitro alkaline elution/rat hepatocyte assay is a sensitive assay for genotoxicity, measured as DNA strand breaks induced in primary cultures of rat hepatocytes after 3-h treatments with test compounds. Since DNA degradation can be rapid and extensive in dead and/or dying cells, the original criteria for a positive result in the assay were that a compound induce a 3.0-fold or greater increase in the elution slope (for the terminal phase of alkaline elution from 3 to 9 h) in the absence of significant cytotoxicity (defined as relative cell viability of less than 70% by trypan blue dye exclusion; TBDE). Recently we have shown that false-positive results can still be obtained due to cytotoxicity when loss of membrane integrity is a late event in toxic cell death relative to the induction of endonucleolytic DNA degradation. To improve the ability of the assay to discriminate between genotoxic vs. cytotoxic effects of chemicals, we have evaluated additional assays of cytotoxicity including cell adenosine triphosphate (ATP) and potassium (K+) content, tetrazolium dye reduction (MTT), TBDE after a further 3-h recovery incubation without test chemicals (delayed toxicity), cell blebbing and endonucleolytic DNA degradation (double-strand breaks; DSBs) assessed by pulsed-field gel electrophoresis (PFGE). We have also evaluated 2 parameters derived from the elution data which can indicate extensive, cytotoxicity-induced DNA degradation: the fraction of the DNA recovered in the neutral lysis/rinse fraction and the gamma-intercept of the extrapolation of the 3-9-h segment of the elution curve. Twenty-eight rodent non-carcinogens that are negative (or inconclusive) in the Ames assay with no, or limited, other evidence of genotoxicity, and 33 genotoxins, most of which are also carcinogens, were evaluated. The results showed that DNA degradation as measured by a 1-h PACE (Programmed Autonomously Controlled Electrodes)/PFGE assay was a sensitive indicator of cytotoxicity which correlated well with results of the other cytotoxicity indicators. The delayed TBDE (after a 3-h recovery), intracellular potassium and ATP assays as well as the gamma-intercept parameter were also shown to be sensitive and in some cases complementary measures of cytotoxicity. Using new criteria based on these data of an induced slope (treatment slope-negative control slope) of 0.020 for the 3- to 9-h elution period and cytotoxicity limits of 70% relative viability for the delayed TBDE assay and 50% for intracellular ATP content, the assay scores the genotoxicity of these 61 reference compounds with an overall accuracy of 92%. Test results using these new criteria are provided for an additional 20 compounds (5 non-genotoxic carcinogens and 15 compounds whose genotoxic and carcinogenic potential are unknown or equivocal).