Genotoxic Hepatocarcinogens Research Articles

Prediction model of potential hepatocarcinogenicity of rat hepatocarcinogens using a large-scale toxicogenomics database

The present study was performed to develop a robust gene-based prediction model for early assessment of potential hepatocarcinogenicity of chemicals in rats by using our toxicogenomics database, TG-GATEs (Genomics-Assisted Toxicity Evaluation System developed by the Toxicogenomics Project in Japan). The positive training set consisted of high- or middle-dose groups that received 6 different non-genotoxic hepatocarcinogens during a 28-day period. The negative training set consisted of high- or middle-dose groups of 54 non-carcinogens. Support vector machine combined with wrapper-type gene selection algorithms was used for modeling. Consequently, our best classifier yielded prediction accuracies for hepatocarcinogenicity of 99% sensitivity and 97% specificity in the training data set, and false positive prediction was almost completely eliminated. Pathway analysis of feature genes revealed that the mitogen-activated protein kinase p38- and phosphatidylinositol-3-kinase-centered interactome and the v-myc myelocytomatosis viral oncogene homolog-centered interactome were the 2 most significant networks. The usefulness and robustness of our predictor were further confirmed in an independent validation data set obtained from the public database. Interestingly, similar positive predictions were obtained in several genotoxic hepatocarcinogens as well as non-genotoxic hepatocarcinogens. These results indicate that the expression profiles of our newly selected candidate biomarker genes might be common characteristics in the early stage of carcinogenesis for both genotoxic and non-genotoxic carcinogens in the rat liver. Our toxicogenomic model might be useful for the prospective screening of hepatocarcinogenicity of compounds and prioritization of compounds for carcinogenicity testing.

Identification of biomarkers of chemically induced hepatocarcinogenesis in rasH2 mice by toxicogenomic analysis

Toxicogenomic approaches have been applied to chemical-induced heptocarcinogenesis rodent models for the identification of biomarkers of early-stage hepatocarcinogenesis and to help clarify the underlying carcinogenic mechanisms in the liver. In this study, we used toxiciogenomic methods to identify candidate biomarker genes associated with hepatocarcinogenesis in rasH2 mice. Blood chemical, histopathologic, and gene expression analyses of the livers of rasH2 mice were performed 7 and 91days after the administration of the genotoxic hepatocarcinogens 2-acetylaminofluorene (AAF) and diethylnitrosoamine (DEN), the genotoxic carcinogen melphalan (Mel), and the nongenotoxic noncarcinogen 1-naphthylisothiocynate (ANIT). Histopathologic lesions and a rise in accompanying serum marker levels were found in the DEN-treated rasH2 mice, whereas no neoplastic lesions were observed in the rasH2 mice. However, biological functional analysis using Ingenuity Pathways Analysis (IPA) software revealed that genes with comparable molecular and cellular functions were similarly deregulated in the AAF- and DEN-treated rasH2 mice. We selected 68 significantly deregulated genes that represented a hepatocarcinogen-specific signature; these genes were commonly deregulated in both the AAF- and DEN-treated rasH2 mice on days 7 and 91. Hierarchical clustering analysis indicated that the expression patterns of the selected genes in the hepatocarcinogen (AAF and DEN) groups were distinctive from the patterns in the control, Mel, and ANIT groups. Biomarker filter analysis using IPA software suggested that 28 of the 68 signature genes represent promising candidate biomarkers of cancer. Quantitative real-time PCR analysis confirmed that the deregulated genes, which exhibited sustained up- and down-regulation up to day 91, are likely involved in early-stage hepatocarcinogenesis. In summary, the common and significant gene expression changes induced by AAF and DEN may reflect early molecular events associated with hepatocarcinogenesis, and these "signature" genes may be useful as biomarkers of hepatocarcinogenesis in mice.

Unique Impact of RB Loss on Hepatic Proliferation: TUMORIGENIC STRESSES UNCOVER DISTINCT PATHWAYS OF CELL CYCLE CONTROL

The retinoblastoma (RB) tumor suppressor pathway is disrupted at high frequency in hepatocellular carcinoma. However, the mechanisms through which RB modulates physiological responses in the liver remain poorly defined. Despite the well established role of RB in cell cycle control, the deletion of RB had no impact on the kinetics of cell cycle entry or the restoration of quiescence during the course of liver regeneration. Although these findings indicated compensatory effects from the RB-related proteins p107 and p130, even the dual deletion of RB with p107 or p130 failed to deregulate hepatic proliferation. Furthermore, although these findings suggested a modest role for the RB-pathway in the context of proliferative control, RB loss had striking effects on response to the genotoxic hepatocarcinogen diethylnitrosamine. With diethylnitrosamine, RB deletion resulted in inappropriate cell cycle entry that facilitated secondary genetic damage and further uncoupling of DNA replication with mitotic entry. Analysis of the mechanism underlying the differential impact of RB status on liver biology revealed that, while liver regeneration is associated with the conventional induction of cyclin D1 expression, the RB-dependent cell cycle entry, occurring with diethylnitrosamine treatment, was independent of cyclin D1 levels and associated with the specific induction of E2F1. Combined, these studies demonstrate that RB loss has disparate effects on the response to unique tumorigenic stresses, which is reflective of distinct mechanisms of cell cycle entry.

RB has a critical role in mediating the in vivo checkpoint response, mitigating secondary DNA damage and suppressing liver tumorigenesis initiated by aflatoxin B1

Hepatocellular carcinoma (HCC) is a significant worldwide health concern that is associated with discrete etiological events, encompassing viral infection, metabolic stress and genotoxic compounds. In particular, exposure to the genotoxic hepatocarcinogen aflatoxin B1 (AFB1) is a significant factor in the genesis of human liver cancer. Presumably, genetic events associated with HCC could influence the effect of environmental insults, yielding a predilection for tumor development. The retinoblastoma (RB) tumor suppressor pathway is functionally inactivated in HCC through discrete mechanisms; however, the role of RB in suppressing tumorigenesis in this disease is poorly understood. Therefore, we analysed how RB status affects the response to AFB1 in reference to acute exposures and tumor development reflective of chronic exposure. Liver-specific Rb deletion resulted in an aberrant proliferative response to AFB1. This cell-cycle induction was associated with increased levels of secondary genetic damage and failure in appropriate cell-cycle coupling. This effect of RB loss was unique to AFB1 and involved the induction of a non-canonical proliferative pathway, and was not merely reflective of the overall cell-cycle deregulation or aberrant regenerative responses. The acute responses to AFB1 exposure presaged aberrations in hepatocyte nuclear morphology and ploidy with RB loss. Correspondingly, RB-deficient livers showed significantly enhanced susceptibility to liver tumorigenesis initiated by AFB1. Combined, these studies show that RB has a critical role in mediating checkpoint responses in liver tissue to maintain genome integrity and in suppressing tumorigenesis.

Dose-dependent alterations in gene expression in mouse liver induced by diethylnitrosamine and ethylnitrosourea and determined by quantitative real-time PCR

We examined the dose-dependency of gene expression changes for 51 genes in mouse liver treated with two N-nitroso genotoxic hepatocarcinogens, diethylnitrosamine (DEN) and ethylnitrosourea (ENU) by quantitative real-time PCR (qPCR). DEN (3, 9, 27 and 80 mg/kg bw) or ENU (6, 17, 50 and 150 mg/kg bw) was injected intraperitoneally into groups of five male 9-week-old B6C3F 1 mice and the livers were dissected after 4 h and 28 days. Total RNA from pooled livers was reverse-transcribed to cDNA and the amount of each gene was quantified by qPCR. Results were analyzed by hierarchical and k-means clustering and ingenuity pathway analysis (IPA). The most characteristic result was a similar dose-dependency of gene expression changes with DEN and ENU. Twenty-one genes exhibited a distinct dose-dependent increase in expression at 4 h for both carcinogens [ Bax, Btg2, Ccng1, Cdkn1a, Cyp4a10, Cyp21a1, Fos, Gadd45b, Gdf15, Hmox1, Hspb1, Isg20l1, Jun, Mbd1, Mdm2, Myc, Net1, Plk2, Ppp1r3c, Rcan1 and Tubb2c], although the increase in gene expression due to ENU was generally weaker than that due to DEN. Only Gdf15 showed a dose-dependent increase in expression at 28 days for both carcinogens. The differences between DEN and ENU were in the expression of additional genes (7 for DEN and 8 for ENU). IPA extracted five gene networks: Network-1 included genes related to cancer and cell cycle arrest and associated with Bax, Btg2, Ccng1, Cdkn1a, Gadd45b, Gdf15, Hspb1, Mdm2 and Plk2 and Network-2 was related to DNA replication, recombination, repair and cell death and associated with Cyp21a1, Gdf15, Ppp1r3c, Rcan1 and Tubb2c. The present results show a distinct dose-dependency of gene expression changes induced by DEN and ENU. These changes were associated with cancer, cell cycle arrest, DNA replication, recombination, repair and cell death and were seen not only at 4 h but also, for some, at 28 days after administration.

Evidence of a Threshold-Effect for 2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline Liver Carcinogenicity in F344/DuCrj Rats

To estimate potential human risk of exposure to a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), a 2-year carcinogenicity test was conducted using male F344 rats administered MeIQx-containing diet at doses of 0 (control), 0.001, 1, and 100 ppm. The lowest dose 0.001 ppm was established as equivalent to the daily intake of this carcinogen in humans (0.2 to 2.6 microg/man/day). Significant decreases of survival rate and body weight gain were observed in rats treated with 100 ppm MeIQx. Histopathological examination revealed significant induction of hepatocellular carcinomas, adenomas, and development of glutathione S-transferase placental form-positive foci with MeIQx at 100 ppm. Moreover, the incidences of Zymbal's glands carcinoma, mammary fibroadenoma, and subcutaneous fibroma were found significantly increased in a 100 ppm MeIQx group. However, no significant induction of altered preneoplastic hepatocellular foci was observed in 0.001 and 1 ppm groups as compared to the controls. 8-Hydroxy-2'-deoxyguanosine levels in the rat liver DNA of the 100 ppm-treated group were not elevated, but MeIQx-DNA adduct formation increased as compared with the 1 ppm case, albeit without significance. No significant induction of any other neoplastic lesions related to the carcinogen administration was found in MeIQx-administered groups except for 100 ppm. These results imply that 1 ppm may be a no-effect level for MeIQx carcinogenesis.

Genetic and epigenetic changes in rat preneoplastic liver tissue induced by 2-acetylaminofluorene

Genotoxic carcinogens, including 2-acetylaminofluorene (2-AAF), in addition to exerting their genotoxic effects, often cause a variety of non-genotoxic alterations in cells. It is believed that these non-genotoxic effects may be indispensable events in tumorigenesis; however, there is insufficient knowledge to clarify the role of carcinogens in both the genetic and epigenetic changes in premalignant tissues and a lack of conclusive information on the link between epigenetic alterations and carcinogenic exposure. In the current study, we investigated whether or not the mechanism of 2-AAF-induced hepatocarcinogenesis consists of both genotoxic (genetic) and non-genotoxic (epigenetic) alterations. Male and female Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 6, 12, 18 or 24 weeks. The levels of DNA adducts obtained from 2-AAF in liver and kidney tissues were assessed by high-performance liquid chromatography combined with electrospray tandem mass spectrometry (HPLC-ES-MS/MS). N-(Deoxyguanosine-8-yl)-2-aminofluorene was the major adduct detected at all time points in both tissues. Global DNA methylation in the livers and kidneys, as determined by an HpaII-based cytosine extension assay and by HPLC-ES-MS/MS, did not change over the 24-week period. In the livers of male rats, there was a progressive decrease of global and long interspersed nucleotide element-1-associated histone H4 lysine 20 trimethylation, as well as hypermethylation of the p16(INK4A) gene. These epigenetic changes were not observed in the livers of female rats or the kidneys of both sexes. Importantly, morphological evidence of formation and progression of neoplastic process was observed in the liver of male rats only. In conclusion, we have demonstrated that exposure of rats to genotoxic hepatocarcinogen 2-AAF, in addition to formation of 2-AAF-specific DNA lesions, resulted in substantial alterations in cellular epigenetic status.

Epigenetic reprogramming of liver cells in tamoxifen‐induced rat hepatocarcinogenesis

Tamoxifen, a nonsteroidal anti-estrogen, is a potent genotoxic hepatocarcinogen in rats, with both tumor initiating and promoting properties. Recently it has been demonstrated that genotoxic carcinogens, in addition to exerting genotoxic effects, often cause epigenetic alterations and these induced epigenetic changes may play important mechanistic role in carcinogenesis. In the present study, we investigated the role of tamoxifen-induced epigenetic changes in hepatocarcinogenic process. The results of the study showed that exposure of female F344 rats to tamoxifen resulted in progressive loss of CpG methylation in regulatory sequences of long interspersed nucleotide elements (LINE-1) and prominent increase in expression of LINE-1 elements and c-myc proto-oncogene. The accumulation of tamoxifen-induced DNA lesions was accompanied by the decreased level of Rad51, Ku70, and DNA polymerase beta (Polbeta) proteins that play a crucial role in maintenance of genomic stability. Furthermore, feeding rats with tamoxifen-containing diet led to increased regenerative cell proliferation, as indicated by the increased level of Ki-67 and proliferating cell nuclear antigen (PCNA) proteins. These data indicate that exposure of animals to genotoxic hepatocarcinogen tamoxifen led to early phenotypical alterations in livers characterized by emergence of epigenetically reprogrammed cells with a specific cancer-related epigenetic phenotype prior to tumor formation.

Comparison of basal gene expression profiles and effects of hepatocarcinogens on gene expression in cultured primary human hepatocytes and HepG2 cells

Toxicogenomics is a relatively new discipline of toxicology. Microarrays and bioinformatics tools are being used successfully to understand the effects of toxicants on in vivo and in vitro model systems, and to gain a better understanding of the relevance of in vitro models commonly used in toxicological studies. In this study, cDNA filter arrays were used to determine the basal expression patterns of human cultured primary hepatocytes from different male donors; compare the gene expression profile of HepG2 to that of primary hepatocytes; and analyze the effects of three genotoxic hepatocarcinogens; aflatoxin B 1 (AFB 1), 2-acetylaminofluorene (2AAF), and dimethylnitrosamine (DMN), as well as one non-gentoxic hepatotoxin, acetaminophen (APAP) on gene expression in both in vitro systems. Real-time PCR was used to verify differential gene expression for selected genes. Of the approximately 31,000 genes screened, 3–6% were expressed in primary hepatocytes cultured on matrigel for 16 h. Of these genes, 867 were expressed in cultured hepatocytes from all donors. HepG2 cells expressed about 98% of the genes detectable in cultured primary hepatocytes, however, 31% of the HepG2 transcriptome was unique to the cell line. A number of these genes are expressed in human liver but expression is apparently lost during culture. There was considerable variability in the response to chemical carcinogen exposure in primary hepatocytes from different donors. The transcription factors, E2F1 and ID1 mRNA were increased three-fold and six-fold ( P<0.05, P<0.01), respectively, in AFB 1 treated primary human hepatocytes but were not altered in HepG2. ID1 expression was also increased by dimethylnitrosamine, acetylaminofluorene and acetaminophen in both primary hepatocytes and HepG2. Identification of genes that are expressed in primary hepatocytes from most donors, as well as those genes with variable expression, will aid in understanding the variability in human reactions to drugs and chemicals. This study suggests that identification of biomarkers of exposure to some chemicals may be possible in the human through microarray analysis, despite the variability in responses.

Characteristic Expression Profiles Induced by Genotoxic Carcinogens in Rat Liver

When applied in toxicological studies, the recently developed gene expression profiling techniques using microarrays, which brought forth the new field of toxicogenomics, facilitate the interpretation of a toxic compound's mechanism of action. In this study, we investigated whether genotoxic carcinogens at doses known to induce liver tumors in the 2-year rat bioassay deregulate a common set of genes in a short-term in vivo study and, if so, whether these deregulated genes represent defined biological pathways. Rats were dosed with the four genotoxic hepatocarcinogens dimethylnitrosamine (4 mg/kg/day), 2-nitrofluorene (44 mg/kg/day), aflatoxin B1 (0.24 mg/kg/day), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 20 mg/kg/day). After treatment for up to 14 days, the expression profiles of the livers were analyzed on Affymetrix RG_U34A microarrays. Among the significantly upregulated genes were a set of target genes of the tumor suppressor protein p53, indicating a DNA damage response. Such a response was expected and, therefore, confirmed the validity of our approach. In addition, the gene expression changes suggest a specific detoxification response, the activation of proliferative and survival signaling pathways, and some cell structural changes. These responses were strong throughout the 14 day time course for 2-nitrofluorene and aflatoxin B1; in the case of dimethylnitrosamine and NNK, the effects were weakly detectable at day 1 and then increased with time. For dimethylnitrosamine and aflatoxin B1, which caused observable inflammation in vivo, we found a corresponding upregulation of inflammatory genes at the same time points. Thus, by the toxicogenomic analysis of short-term in vivo studies, we identified genes and pathways commonly deregulated by genotoxic carcinogens, which may be indicative for the early events in tumorigenesis and, thus, predictive of later tumor development.

Strong induction of p73 protein in vivo coincides with the onset of apoptosis in rat liver after treatment with the hepatocarcinogen N-nitrosomorpholine (NNM).

Treatment of rats with genotoxic hepatocarcinogens such as N-nitrosomorpholine (NNM) causes severe hepatotoxicity associated with apoptosis of hepatocytes beginning after 12 h. Previously, we reported that after a single administration of high NNM dose p53 protein level increased in liver but not in testis and that the first wave of apoptosis preceded the induction of p53 indicating that apoptosis in liver was driven by a p53-independent pathway. We now show a pronounced upregulation of p73 protein, a p53-related gene product. The increase of p73 alpha and beta occurred already 6 h after NNM administration and preceded the onset of apoptosis by 6 h. Very strong p73 signals appeared 20 and 40 h post-treatment and persisted for a few days, whereas p53 was induced only transiently at 20 and 40 h post-treatment. Immunohistochemical analysis revealed that unlike p53, p73 was detected in the nuclei of hepatocytes undergoing apoptosis. Following the upregulation of p73 levels, the products of several genes regulating DNA repair, e.g., GADD-45 and p53R2 and mediating apoptosis such as apoptosis inducing factor (AIF) were rapidly induced, whereas transient elevation of MDM-2 protein was delayed and coincided temporary with activation of p53 protein. Interestingly, NF-kappaB another transcription factor responding to cellular stress was activated at 20 h after NNM administration and reached a maximum after an additional 20 h. Our data indicate that activated p73 protein may positively affect the induction and execution of apoptosis in response to genotoxic action of NNM.

Riddelliine N-oxide is a phytochemical and mammalian metabolite with genotoxic activity that is comparable to the parent pyrrolizidine alkaloid riddelliine

Pyrrolizidine alkaloids (PAs) and their N-oxide derivatives are naturally-formed genotoxic phytochemicals that are widely distributed throughout the world. Although, the quantities of PAs and PA N-oxides in plants are nearly equal, the biological and genotoxic activities of PA N-oxides have not been studied extensively. PA N-oxides are major metabolites of PAs and are generally regarded as detoxification products. However, in this study, we report that rat liver microsomes converted riddelliine N-oxide to the genotoxic 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) metabolite. Metabolism of riddelliine N-oxide by rat liver microsomes under hypoxic conditions (argon) generated predominantly the parent PA, riddelliine. The reduction of riddelliine N-oxide to riddelliine was diminished, when the metabolism of riddelliine N-oxide with rat liver microsomes was conducted aerobically. Rat liver microsomal incubations of riddelliine N-oxide in the presence of calf thymus DNA produced a set of DHP-derived DNA adducts as detected and quantified by 32 P -postlabeling/HPLC. The same DHP-derived DNA adducts were also found in liver DNA of F344 rats fed riddelliine N-oxide or riddelliine. When rats received doses of 1.0 mg/kg riddelliine N-oxide for three consecutive days, the level of DNA adducts was 39.9±0.6 adducts/10 7 nucleotides, which was 2.6-fold less than that measured in rats treated with riddelliine at the same dose. We have previously shown that these DHP-derived DNA adducts are produced by chronic feeding of riddelliine and that the adduct levels correlated with liver tumor formation. Results presented in this paper indicate that riddelliine N-oxide, through its conversion to riddelliine, is also a potential genotoxic hepatocarcinogen.

Lack of initiation activity in rat liver of low doses of 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline

It has been generally accepted that genotoxic carcinogens have no threshold in exerting their potential for cancer induction. However, the non-threshold theory can be challenged for cancer risk assessment in humans. Here we examined low dose carcinogenicity of a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx), using an in vivo medium-term bioassay to detect initiating activity for rat hepatocarcinogenesis. With MeIQx initiation at various doses followed by administration of phenobarbital, a well known hepatopromoter, no induction of glutathione S-transferase placental form-positive foci, assessed as preneoplastic lesions, was noted at doses of 0.001–1 ppm. The results imply a no-observed effect level for hepatocarcinogenicity with this genotoxic agent.

Value of GST-P Positive Preneoplastic Hepatic Foci in Dose-Response Studies of Hepatocarcinogenesis: Evidence for Practical Thresholds with Both Genotoxic and Nongenotoxic Carcinogens. A Review of Recent Work

Recent low-dose carcinogenesis studies, including major group projects are reviewed. The prevailing paradigm is that carcinogens, particularly genotoxic compounds, have no threshold in exerting their potential for cancer induction. However, the nonthreshold hypothesis can be challenged for cancer risk assessment in humans. A recent very large-scale cooperative effort in Japan furthermore showed that the genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, forms DNA adducts and 8-hydroxy-2'-deoxyguanosine at low doses, but does not induce glutathione S-transferase placental form (GST-P) positive foci as preneoplastic lesions in rat liver (< or = 10 ppm in diet). Moreover, very low doses of a N-nitroso compound. diethylnitrosamine (DEN), were also found not to significantly induce GST-P positive foci in rat liver (< or = 0.01 ppm in drinking water). Given the direct correlation between induction of the preneoplastic lesions in the short-term and carcinomas in the longer term with different carcinogens, the results imply a practical nonobserved effect level for hepatocarcinogenicity. Similar results were also observed with so-called nongenotoxic carcinogens such as phenobarbital (PB) and p,p-dichlorodiphenyltrichloroethane (DDT), which do not exert positive effects on lesion development at very low doses. Furthermore, experiments with application of PB and DDT after treatment with DEN indicate that at very low doses (< or = 2 ppm in diet), they may even inhibit the development of GST-P positive foci. The data reviewed provide evidence that preneoplastic foci in the liver can be employed as end-point lesions in place of tumors and that exposure to very low levels of carcinogens, typical of those found in the human environment, does not necessarily present as a risk factor.

Lack of a dose-response relationship for carcinogenicity in the rat liver with low doses of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline or N-nitrosodiethylamine.

For a long period, it has been generally considered that carcinogens, particularly genotoxic ones, have no threshold in exerting their potential for cancer induction. However, the non‐threshold theory can be challenged with regard to assessment of cancer risk to humans. Here we show that a food‐derived, genotoxic hepatocarcinogen, 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline, forms DNA adducts at low doses, but does not induce glutathione S‐transferase placental form (GST‐P)‐positive foci (considered to be preneoplastic lesions) or 8‐hydroxy‐2′‐deoxyguanosine in rat liver. Moreover a N‐nitroso compound, N‐nitrosodiethylamine, at low doses was also found not to induce GST‐P‐positive foci in rat liver. These results imply that there is a no‐observed effect level for hepatocarcinogenesis by these genotoxic carcinogens.

Distinction of carcinogens from mutagens by induction of liver cell foci in a model for detection of initiation activity

Initiating activities of 26 chemicals were investigated in an in vivo 5 week initiation assay model with evaluation of the induction of glutathione S-transferase placental form (GST-P) positive foci as end-point lesions. With the five genotoxic hepatocarcinogens (diethylnitrosamine, dimethylnitrosamine, 2-acetylaminofluorene, N-bis(2-hydroxypropyl)-nitrosamine and safrole) and 11 genotoxic non-hepatocarcinogens, (2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, benzo[ a]pyrene, N-butyl- N-(4-hydroxybutyl)nitrosamine, 7,12-dimethylbenz[ a]anthracene, 1,2-dimethylhydrazine, N-ethyl- N-hydroxyethylnitrosamine, 3-methylcholanthrene, N-methyl- N-nitrosourea, N-methyl- N′-nitro- N-nitrosoguanidine, 4-nitroquinoline 1-oxide and 8-hydroxyquinoline), the numbers of GST-P positive foci were significantly higher than in the controls. On the other hand, the mutagenic non-carcinogens (quercetin, p-phenylenediamine dihydrochloride, 2-chloroethanol and 6-hydroquinoline) did not cause a significant increase. Similarly, non-genotoxic hepatocarcinogens of the hepatopromotor class and promotors which target organs other than the liver did not induce GST-P positive foci. The specificity was thus remarkable. Moreover, regardless of the target organ, mutagenic carcinogens were detected by this in vivo 5 week initiation assay, which therefore constitutes a powerful method for screening for carcinogenic potential, especially in the initiation stage of carcinogenesis.

Role of transforming growth factor alpha and prostaglandins in preferential growth of preneoplastic rat hepatocytes

The role of transforming growth factor alpha (TGFalpha) and prostaglandins (PGs) in the preferential growth of preneoplastic liver cells was studied. Rats received the genotoxic hepatocarcinogen N-nitrosomorpholine (NNM); placental glutathione S-transferase (GSTp) was used as a marker to identify preneoplastic foci. Preneoplastic foci expressing TGFalpha (TGFalpha(+)) grew more rapidly than TGFalpha negative (TGFalpha(-)) ones. Almost all tumours studied were positive for TGFalpha. The key enzymes of prostaglandin synthesis, cyclooxygenase I (Cox-1) and II (Cox-2), were present in all unaltered and preneoplastic cells and tended to decrease in the later stages of hepatocarcinogenesis. Immunostaining revealed that cultures of hepatocytes, isolated from NNM-treated livers by collagenase perfusion, contained 1-2% GSTp-positive (GSTp(+)) and 9% TGFalpha(+) hepatocytes; 0.6% of the cells were GSTp(+)/TGFalpha(+). Cox-1 and Cox-2 were present in all cells. DNA replication was almost exclusively associated with expression of TGFalpha. GSTp(+) hepatocytes showed a 3- to 4-fold higher probability of TGFalpha expression and of DNA synthesis than GSTp-negative (GSTp(-)) cells. PGE(2) or PGF(2alpha) increased expression of TGFalpha and DNA replication in GSTp(-) cells but not in GSTp(+) cells. PGA(2) and PGJ(2) decreased DNA synthesis in TGFalpha(+) cells without an obvious effect on the intracellular levels of TGFalpha. The Cox-2 inhibitor SC236 suppressed DNA replication preferentially in GSTp(+) cells; this inhibition was reversed by PGE(2)/F(2alpha). Indomethacin had no effect. These results suggest the following conclusions. (i) Growth regulation of preneoplastic GSTp(+) cells in culture exhibits distinct differences from GSTp(-) cells and elevated expression of TGFalpha contributes to their growth advantage. (ii) TGFalpha renders preneoplastic hepatocytes sensitive to suppression of DNA synthesis by PGA(2)/J(2). (iii) SC236, a Cox-2 inhibitor, may have preventive value in hepatocarcinogenesis.

Induction of DNA synthesis in mouse liver following increases of DNA adduct levels elicited by very low cumulative doses of the genotoxic hepatocarcinogen 7H-dibenzo[c,g]carbazole.

The purpose of this work was to investigate the administration of very low but repeated doses of a genotoxic carcinogen and an eventual correlation with cellular DNA synthesis. The compound 7H-dibenzo[c,g]carbazole is a genotoxic carcinogen in the mouse liver and was administered topically at the dose of 13.35 microg per animal every 2 days to give a total of 13 applications. Animals were sacrificed 48 hours after every 2 applications until the 10th treatment, then 48 hours after every treatment. Postulated genotoxic effects such as DNA adduct formation were detected by the 32P-post labeling assay. Liver sections were examined for microscopic changes and DNA synthesis. Results showed an increase of the total DNA adduct level in the liver throughout the study with a slowing down in the level after the sixth application of the compound. This change could correspond to the onset of DNA synthesis and to the moderate hepatocellular apoptosis which was observed. The DNA synthesis, which was considered to be secondary to the cytotoxicity induced by the high level of DNA adducts altering normal cellular activity, could also be the opportunity to fix the DNA adducts into heritable mutations. These results raise the question regarding the risk assessment in humans exposed regularly to very low doses of chemicals in the environment: for non-proliferating tissue, the regular accumulation of DNA adducts could remain silent until a "threshold level" is reached from which stimulation of the DNA synthesis may fix the DNA adducts into heritable mutations, eventually leading to tumors.

Initiated rat hepatocytes in primary culture: a novel tool to study alterations in growth control during the first stage of carcinogenesis.

To study growth regulation in the beginning of carcinogenesis, we established a novel ex vivo model for co-cultivation of normal and putatively initiated hepatocytes. Rats received the genotoxic hepatocarcinogen N-nitrosomorpholine (NNM). This led to the appearance of hepatocytes expressing placental glutathione S-transferase (G(+) cells). These cells exhibited elevated rates of cell replication and apoptosis, as known from further advanced preneoplasia; G(+) cells were considered initiated. At days 20-22 post-NNM treatment their frequency was maximal (1-2%); approximately 40% were still single and 60% were arranged in mini foci. At this time-point liver cells were isolated by collagenase perfusion and cultivated. G(+) cells, identified by immunostaining of the culture-plates, were present at the same percentage as in vivo, excluding selective loss, enrichment or spontaneous expression of the G(+) phenotype. In untreated cultures G(+) hepatocytes showed significantly higher rates of replicative DNA synthesis than normal G(-) cells. Application of the hepatomitogen cyproterone acetate (CPA) elevated DNA replication preferentially in G(+) cells. Transforming growth factor beta1 (TGF-beta1) suppressed replicative DNA synthesis which was more pronounced in G(+) than in G(-) hepatocytes. Combined treatment with CPA and TGF-beta1 had no effect on G- cells, but considerably inhibited DNA replication in G(+) cells. This suggests that the effects of TGF-beta1 predominated in G(+) hepatocytes. We conclude that putatively initiated G(+) hepatocytes, both in vivo and in culture, exhibit higher basal rates of DNA replication than normal G(-) hepatocytes and an over-response to mitogens and growth inhibitors. Therefore, G(+) cells show (i) nearly identical behaviour in intact liver and in primary culture and (ii) inherent defects in growth control that are principally similar although somewhat less pronounced than in later stages of carcinogenesis. The present ex vivo system thus provides a novel and useful tool to elucidate biological and molecular changes during initiation of carcinogenesis.

Effect of mitogenic or regenerative cell proliferation on lacz mutant frequency in the liver of MutaTMMice treated with 5, 9-dimethyldibenzo[c,g]carbazole.

The purpose of this work was to investigate the impact of cell proliferation on liver mutagenesis. The genotoxic hepatocarcinogen 5, 9-dimethyldibenzo[c,g]carbazole (DMDBC) was administered to lacZ transgenic MutaTMMice at a non-hepatotoxic dose of 10 mg/kg, which induces only a slight increase in the liver lacZ mutant frequency (MF). To determine if cell proliferation stimuli enhanced DMDBC mutagenicity, MF was analyzed in mice first receiving DMDBC 10 mg/kg, then approximately 2 weeks later, either carbon tetrachloride (CCl4, a cytotoxic agent inducing regenerative cell proliferation) or phenobarbital (PB, a mitogenic agent inducing direct hyperplasia). In preliminary studies, the extent of cell proliferation induced by CCl4, PB and DMDBC was determined in non-transgenic CD2F1 mice by means of 5-bromodeoxyuridine labeling. The labeling index was significantly increased after CCl4 and PB, while no change was detected with DMDBC. MF was then determined in MutaTMMice 28 days after initial DMDBC treatment. No increase in MF was detected in mice receiving CCl4 or PB alone. A 2- to 3-fold increase in MF was detected in mice treated with 10 mg/kg DMDBC alone. In contrast, MF was markedly increased in mice receiving DMDBC followed by proliferative treatment (15-fold with CCl4 and 25-fold with PB). These results demonstrate that expression of DMDBC-induced mutations in mouse liver largely depends on the induction of cell proliferation (by a cytotoxic or mitogenic stimulus) and illustrate that MutaTMMouse is a valuable tool to investigate the early events of liver carcinogenesis.