Hepatocarcinogenic Potential Research Articles

Gene expression profiling in the liver and lung of perfluorooctane sulfonate-exposed mouse fetuses: Comparison to changes induced by exposure to perfluorooctanoic acid

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are environmental contaminants found in the tissues of humans and wildlife. They are activators of peroxisome proliferator-activated receptor-alpha (PPARα) and exhibit hepatocarcinogenic potential in rats. PFOS and PFOA are also developmental toxicants in rodents and PFOS has been shown to induce pulmonary deficits in rat offspring. Pregnant CD-1 mice were dosed with 0, 5, or 10mg/kg PFOS from gestation days 1–17. Transcript profiling was conducted on the fetal liver and lung. Results were contrasted to data derived from a previous PFOA study. PFOS-dependent changes were primarily related to activation of PPARα. No remarkable differences were found between PFOS and PFOA. Given that PPARα signaling is required for neonatal mortality in PFOA-treated mice but not those exposed to PFOS, the neonatal mortality observed for PFOS may reflect functional deficits related to the physical properties of the chemical rather than to transcript alterations.

Safrole-DNA adduct in hepatocellular carcinoma associated with betel quid chewing

Betel quid chewing, which contributes high concentration of safrole in saliva, is a popular oral habit in Taiwan. Safrole is a documented rodent hepatocarcinogen, yet its hepatocarcinogenic potential in human is not known. Here, we used LC/ESI-ITMS n and LC/QTOF-MS confirmed safrole-dGMP as reference standard to detect the safrole-DNA adduct in hepatic tissues from HBsAg-/HCV-seronegative hepatocellular carcinoma patients by 32P-postlabeling. We first synthesized and confirmed safrole-dGMP by LC/MS. Two isomeric safrole-dGMPs were characterized as N 2-( trans-isosafrol-3′-yl) deoxyguanosine and N 2-(safrol-1′-yl) deoxyguanosine. This technique was able to detect hepatic safrole-DNA adduct in mice that were treated with safrole but not sensitive enough to detect safrole-DNA adduct in human samples. Using the nuclease P1 version of the 32P-postlabeling technique, we detected the presence of safrole-DNA adduct in two out of 28 hepatic tissues from hepatocellular carcinoma patients, and only these two patients had a history of betel quid chewing lasting more than 10 years. From co-chromatography with the mass confirmed safrole-dGMPs, this safrole-DNA adduct was identified as N 2-( trans-isosafrol-3′-yl) deoxyguanosine. These results suggest that betel quid-containing safrole might be involved in the pathogenesis of hepatocellular carcinoma in human beings and LC/MS has the potential to identify DNA adducts in clinical samples.

Alternative Mechanism for PFOA?: Trout Studies Shed Light on Liver Effects

Perfluorooctanoic acid (PFOA), used to make a class of industrial chemicals that are widely used in products such as textile coatings and flame retardants, is known to be a potent hepatocarcinogen in rodents. Until now the only mechanism of action for PFOA identified in rodent studies has been peroxisome proliferation, a well-characterized form of oxidative stress. Humans are reportedly insensitive to peroxisome proliferation; however, concerns remain that PFOA may cause adverse effects in people as well as in laboratory animals. Using rainbow trout as a model for chemically induced liver cancer in humans, a team of researchers suggest a new mechanism for the carcinogenicity of PFOA that does not involve peroxisome proliferation [EHP 116:1047–1055; Tilton et al.]. The investigators compared the hepatocarcinogenic potential of PFOA against the structurally diverse peroxisome proliferators cloribrate (CLOF) and dehydroepiandrosterone (DHEA), identifying mechanisms of carcinogenesis from hepatic gene expression profiles phenotypically anchored to tumor outcome. Trout were fed PFOA in the diet for 30 weeks for tumor analysis. The investigators subsequently examined gene expression by cDNA array in animals fed PFOA, DHEA, CHOF, or 17β-estradiol (E2; a known tumor promoter) in the diet for 14 days. The study showed that PFOA and DHEA treatments significantly increased liver tumor incidence and multiplicity; CLOF showed no effect. However, carcinogenesis was independent of peroxisome proliferation as measured by the lack of peroxisomal β-oxidation and catalase activity. On the contrary, both PFOA and DHEA resulted in estrogenic gene signatures closely resembling that of E2. CLOF, however, regulated no genes in common with E2. Although the current study did not identify a threshold for the estrogenic effect of PFOA, the results indicate that PFOA is weakly estrogenic in trout and that its association with trout liver cancer may be related to disruptions in estrogenic signaling. More studies are needed to assess the potential for PFOA-mediated carcinogenesis in other species that are insensitive to peroxisome proliferation. Considering the mechanism identified in this study, the consequences of hormone-related effects by PFOA should be evaluated in other tissues, models, and sensitive life stages.

Hepatitis B virus infection: the burden of disease in South Africa

Hepatitis B virus infection, both acute and chronic, occurs commonly in the black population of South Africa, and chronic infection and its sequelae of cirrhosis and hepatocellular carcinoma are major public health threats. Chronic hepatitis B virus infection is rare in the other population groups, with the exception of the very small Chinese community. Prevalences of chronic carriage of hepatitis B virus in South African blacks are 5-16% in rural males, 8-9% in urban males, 4-12% in rural females, and 2.7-4% in urban females. The overall male to female ratio is 2.6:1.0. There are now three to four million South African blacks who are chronically infected with this virus. In rural black populations chronic hepatitis B virus infection is acquired very early in life, predominantly as a result of horizontal transmission of the virus, and by the age of 5 years carrier rates approach those seen in adulthood. A further slight increase occurs at school-going age and a greater increase at the time of becoming sexually active. Urban black carrier rates are significantly lower and the infection is acquired later in life. The decreased urban viral carriage rates occur mainly in the first generation born in an urban environment. Hepatitis B virus accounts for about 60% of clinically evident acute viral hepatitis among blacks and about 10% of chronic hepatitis and cirrhosis. It is the cause of the majority of the many cases of hepatocellular carcinoma that occur in black South Africans. The tumour is more common in males and in rural-born than in urban-born blacks. The close association between chronic hepatitis B virus infection and hepatocellular carcinoma holds true in rural and urban patients and males and females. The association is age-related, being closer in younger patients. Genotypes A and D of hepatitis B virus predominate in South African isolates, with genotype A and its subtype Aa having a particularly high hepatocarcinogenic potential.

β-Naphthoflavone enhances oxidative stress responses and the induction of preneoplastic lesions in a diethylnitrosamine-initiated hepatocarcinogenesis model in partially hepatectomized rats

The tumour-promoting effects of β-naphthoflavone (BNF), a novel aryl hydrocarbon receptor (AhR) agonist, were investigated using a medium-term hepatocarcinogenesis model in rats. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) at a dose of 200 mg/kg body weight and were fed a diet containing 0% (basal diet), 0.5% or 1% BNF for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after the BNF treatment. The number and area of glutathione S-transferase placental form (GST-P) positive foci significantly increased in the livers of rats treated with BNF with concomitantly increased cell proliferation compared to those in the livers of the DEN alone group. Global gene expression analysis and subsequent quantitative real-time reverse transcription-polymerase chain reaction revealed that BNF induced not only the ‘AhR gene battery’ Cyp1a1, Cyp1a2, Cyp1b1, Nqo1, Aldh3a1 and Ugt1a6 but also the transcription factor NF-E2-related factor 2 (Nrf2)-regulated genes such as Gstm1, Gpx2, Akr7a3 and Yc2 (and also Nqo1), presumably due to the adaptive response against BNF-triggered oxidative stress responses. Reactive oxygen species production increased in microsomes isolated from the livers of BNF-treated rats, and this enhancement was suppressed by the P450 inhibitor SKF-525A. Furthermore, BNF enhanced oxidative DNA damage and lipid peroxidation, estimated by the levels of 8-hydroxydeoxyguanosine (8-OHdG) and thiobarbituric acid-reactive substances. These results suggest that the administration of BNF at a high dose and over a long-term enhance oxidative stress responses which may contribute to its hepatocarcinogenic potential in rats.

Diethylnitrosamine Initiation Does Not Alter Clofibric Acid–Induced Hepatocarcinogenesis in the Rat

Clofibric acid (CLO) is a nongenotoxic hepatocarcinogen in rodents that causes altered hepatocellular foci and/or neoplasms. Initiation by DNA-damaging agents such as diethylnitrosamine (DEN) accelerates focus and tumor appearance and could therefore significantly contribute to shortening of the regulatory 2-year rodent carcinogenicity bioassays. However, it is crucial to evaluate the histological and molecular impact of initiation with DEN on hepatocarcinogenesis promoted by CLO. Male F344 rats were given a single nonnecrogenic injection of DEN (0 or 30 mg/kg) followed by Control diet or CLO (5000 ppm) in diet for up to 20 months. Histopathology and gene expression profiling were performed in liver tumors and surrounding nontumoral liver tissues. The molecular signature of DEN was characterized and its histopathological and immunohistopathological effects on focus and tumor types were also determined. Although foci and tumors appeared earlier in the DEN+CLO-treated group compared to the group treated with CLO alone, DEN had little impact on gene expression in nontumoral tissues since the gene expression profiles were highly similar between Control and DEN-treated rats, and DEN+CLO- and CLO-treated rats. Finally, tumors obtained from DEN+CLO and CLO-treated groups displayed highly correlated gene expression profiles (r>0.83, independently of the time-point). The pathways involved in tumor development revealed by Gene Ontology functional analysis are similar when driven either by spontaneous initiation or by a chemically induced initiation step. Our work described here may contribute to the design optimization of shorter preclinical tests for the evaluation of the nongenotoxic hepatocarcinogenic potential of drugs under development.

Hepatocellular neoplasms after intrahepatic transplantation of ovarian fragments into ovariectomized rats†

Intrahepatic transplantation of ovarian fragments in ovariectomized rats results in morphological abnormalities. The liver acini draining blood from ovarian grafts show alterations resembling chemically induced amphophilic hepatocellular preneoplasias. We investigated the long-term development of these estrogen-induced foci of altered hepatocytes (FAH). We divided 451 Lewis rats into one main group (MG) and 11 (7 female, 4 male) control groups and observed them for up to 30 months. MG animals were ovariectomized and received ovarian transplants into the right liver part. Different combinations of castration, transplantation of ovarian or testicular fragments, and administration of antiestrogenic toremifene were used in controls. In the MG, transplants showed signs of gonadotropic stimulation, and estrogen levels were strongly increased in the downstream liver acini. After 6 and 12 months, FAH developed in hepatocytes downstream of the transplants. After 18 months, 27% of the MG animals showed transformation of FAH into hepatocellular adenomas; this figure increased to 42% after 24 months (8/19), significantly outnumbering four spontaneous adenomas that developed between 18 and 30 months in 258 control animals. Hepatocellular carcinoma (HCC) appeared only in the MG. At 24 and 30 months, 18 HCCs developed; thus, 78% of MG animals showed at least one carcinoma. Administration of toremifene in ovariectomized and transplanted animals completely prevented hepatocarcinogenesis. Testicular grafts showed no influence on liver tissue. In conclusion, initially adaptive but preneoplastic alterations in hepatocytes downstream of intrahepatically transplanted ovarian fragments may transform into HCC, indicating a strong hepatocarcinogenic potential of high local levels of endogenous estrogens in the rat liver.

Increased hepatocarcinogenic potential of hepatitis B virus genotype A in Bantu‐speaking sub‐saharan Africans

Genotypes A, D, and E of the hepatitis B virus (HBV) circulate in southern Africa, with genotype A predominating. Their hepatocarcinogenic potential in Bantu-speaking sub-saharan Africans is, however, unknown. Using a case/control format, we investigated the hepatocarcinogenic potential of these genotypes and subgenotype A1 of genotype A, which accounts for the great majority of genotype A isolates. HBV isolates from 111 unselected Bantu-speaking sub-saharan Africans with hepatocellular carcinoma (HCC) and 111 matched asymptomatic chronic carriers, serving as controls, were genotyped using the method of [Lindh et al. (1997): J Infect Dis 175:1285-1293]. Subgenotypes of genotype A were determined using an in-house restriction fragment length polymorphism assay. Genotype A was present in 96 (86.5%) of patients with HCC and 76 (68.5%) of the carriers, giving a 4.5-fold (95% confidence limits: 1.86, 10.90) increased risk of HCC in carriers infected with genotype A compared with those infected with non-A genotypes. HCC patients infected with genotype A were significantly younger than those infected with non-A genotypes (P = 0.02). The distributions between these two groups according to sex, geographic background, and tribe were not significantly different. Subgenotype A1 was present in all of the 77 cancer patients and 69 of 70 carriers analyzed, yielding a relative risk of 4.21 (95% confidence limits: 1.73,10.23) of HCC in those infected with subgenotype A1 compared with those infected with non-A genotypes. Genotype A has a greater hepatocarcinogenic potential than non-A genotypes in Bantu-speaking sub-saharan Africans and this is entirely attributable to subgenotype A1.

Induction of Hepatocellular Proliferative Lesions in CBA Mice by a 26-week Dietary Administration of Kojic Acid

In our previous study in which diets containing 0, 1.5 or 3% Kojic acid (KA) were administered to heterozygous p53-deficient mice of the CBA strain [p53 (+/-) mice] and their wild-type littermates [p53 (+/+) mice] for 26 weeks, the incidences of hepatocellular adenomas as well as altered hepatocellular foci were increased in p53 (+/-) and p53 (+/+) mice of the KA treated groups without initiation, as compared to those in the untreated control mice. In order to confirm the reproducibility of the induction of hepatocellular proliferative lesions in p53 (+/+) mice given KA, male CBA mice were given dietary administration of 0, 0.5, 1 or 2% KA for 26 weeks. Body weight gain in the 2% KA group was depressed after week 20 as compared to the corresponding control group. Significant increases of absolute and relative liver weights were observed in the groups treated with 1% and 2% KA. The incidences of hepatocellular adenomas in the control and 0.5, 1 and 2% KA groups were 5, 17, 10 and 21%, respectively, and those of hepatocellular foci in these groups were 15, 39, 45 and 47%, respectively, the difference between the control and 2% KA groups being statistically significant. The mean values for multiplicity of hepatocellular adenomas in the control and 0.5, 1 and 2% KA groups were 0.05, 0.2, 0.2 and 0.2, respectively, and those for hepatocellular foci were 0.2, 0.6, 0.8 and 0.6, respectively. The results of the present study suggest that KA has a hepatocarcinogenic potential in CBA mice.

Lack of elevated liver carcinogenicity of aminophenylnorharman in p53-deficient mice

The hepatocarcinogenic potential of 9-(4′-aminophenyl)-9 H-pyrido[3,4- b]indole (aminophenylnorharman, APNH) was investigated using male and female p53 deficient mice. Incidence of oval cell hyperplasia was 2/14 (14.3%), 14/23 (60.9%), and 2/10 (20%) in p53 nullizygous (−/−), heterozygous (+/−), and wild type (+/+) mice, respectively, exposed to 30 ppm APNH for 15 weeks, while hepatocellular anisonucleosis was observed only in APNH-treated p53 (−/−) mice. At 40 weeks, hepatocellular carcinomas had developed in 16/46 (34.8%) and 10/27 (37.0%) of female p53 (+/−) and (+/+) mice in contrast to only 1/45 (2.2%) and 2/12 (16.7%) in their male counterparts, respectively, without any detectable p53 gene mutations. Dose-dependent APNH–DNA adduct formation and transcriptional induction of CYP 1A1, but not CYP 1A2, was revealed with 7-day APNH treatment using female C57BL/6J mice. These results suggested hepatocarcinogenicity of APNH in mice could be linked to the liver microenvironment including hormonal milieu but independent of p53 expression and p53 gene mutations.

Comparison of oxidative stress and changes of xenobiotic metabolizing enzymes induced by phthalates in rats

Phthalates are widely used as a plasticizer and cause a peroxisome proliferation. Peroxisome proliferators (PPs), such as di-2-ethylhexyl phthalate (DEHP) and clofibrate (CF) are known to have a hepatocarcinogenic potential in rodents. It has been proposed that these PPs may cause hepatocellular cancer by an oxidative damage-mediated mechanism(s). The primary purpose of this study is to find whether there is a difference between the oxidative damage by hepatocarcinogenic PPs (DEHP and CF) and the oxidative damage by weak PPs [di-n-butyl phthalate (DBP) and n-butylbenzyl phthalate (BBP)]. The second purpose is to investigate if phthalates can affect the phase I/phase II enzymes, and if the effect of PPs on metabolizing enzymes correlates with peroxisome proliferation or not. After rats were treated with PPs (DEHP, DBP and BBP; 50, 200, 1000 mg/kg, CF; 100 mg/kg, p.o., for 14 days), the activities of metabolizing enzymes and peroxisomal enzymes were investigated, and the oxidative damage was measured using 8-hydroxydeoxyguanosine (8-OHdG) in the DNA and malonedialdehyde (MDA) in the livers. These four PPs significantly increased the relative liver weights, palmitoyl-CoA oxidation and activity of carnitine acetyltransferase. DEHP was found to be the most potent PP among three phthalates. A dramatic and dose-dependent increase in hepatic MDA levels was observed in CF (100 mg/kg), DEHP (⩾50 mg/kg), DBP and BBP (⩾200 mg/kg) groups. However, the 8-OHdG in hepatic DNA was increased only in DEHP (1000 mg/kg) and CF groups. Activities of cytochrome P4501A1, 1A2, 3A4, UDP-glucuronosyl transferase and glutathione S-transferase were decreased overall by PPs, but there is no correlation between the inhibitory effect on metabolizing enzymes and the peroxisome proliferation. These results indicate that 8-OHdG positively correlates with carcinogenic potential of PPs, but other factors as well as peroxisomal H2O2 could be involved in the generation of 8-OHdG and the carcinogenesis of PPs. The present findings also demonstrate that the effect of PPs on xenobiotic metabolizing enzymes may be independent of the peroxisome proliferation and the oxidative stress. Thus it is possible that the PPs affect the hepatic toxification/detoxification capacity even in humans.

Oxidative DNA damage by an N-hydroxy metabolite of the mutagenic compound formed from norharman and aniline

Norharman (9 H-pyrido[3,4- b]indole), which is a heterocyclic amine included in cigarette smoke or cooked foodstuffs, is not mutagenic itself. However, norharman reacts with non-mutagenic aniline to form mutagenic aminophenylnorharman (APNH), of which DNA adducts formation and hepatocarcinogenic potential are pointed out. We investigated whether N-OH-APNH, an N-hydroxy metabolite of APNH, can cause oxidative DNA damage or not, using 32 P -labeled DNA fragments. N-OH-APNH caused Cu(II)-mediated DNA damage. When an endogenous reductant, β-nicotinamide adenine dinucleotide (NADH) was added, the DNA damage was greatly enhanced. Catalase and a Cu(I)-specific chelator inhibited DNA damage, suggesting the involvement of H 2O 2 and Cu(I). Typical OH scavenger did not inhibit DNA damage. These results suggest that the main reactive species are probably copper-hydroperoxo complexes with DNA. We also measured 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) formation by N-OH-APNH in the presence of Cu(II), using an electrochemical detector coupled to a high-pressure liquid chromatograph. Addition of NADH greatly enhanced 8-oxodG formation. UV–VIS spectra and mass spectra suggested that N-OH-APNH was autoxidized to nitrosophenylnorharman (NO-PNH). We speculated that NO-PNH was reduced by NADH. Cu(II) facilitated the redox cycle. In the presence of NADH and Cu(II), very low concentrations of N-OH-APNH could induce DNA damage via redox reactions. We conclude that oxidative DNA damage, in addition to DNA adduct formation, may play an important role in the expression of genotoxicity of APNH.

Induction of liver preneoplastic lesions by aminophenylnorharman, formed from norharman and aniline, in male F344 rats

9-(4′-Aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), produced by the reaction of norharman with aniline in the presence of S9 mix, is a novel heterocyclic amine (HCA), with mutagenicity to Salmonella typhimurium TA 98 and YG 1024 comparable to that of other HCAs such as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). This experiment was designed to investigate its potential to induce glutathione S-transferase placental form (GST-P) positive foci in the liver. Male F344 rats, 7 weeks old, were fed diet containing 0, 10, 20, or 50 ppm APNH for 4 weeks, killed by ether euthanasia and performed complete necropsy. Numbers of GST-P positive foci larger than 0.1 mm in diameter induced by APNH at the dose of 10, 20, and 50 ppm were increased in a dose dependent manner to 0.52, 1.3, and 21 foci/cm2, respectively, with areas of 0.006, 0.01, and 2.3 mm2/cm2. No such GST-P positive foci were observed in rats fed control diet. These findings suggest that APNH has hepatocarcinogenic potential in male F344 rats.

Comparison of the Effects of Various Peroxisome Proliferators on Peroxisomal Enzyme Activities, DNA Synthesis, and Apoptosis in Rat and Human Hepatocyte Cultures

Peroxisome proliferators (PPs) are a class of rodent nongenotoxic hepatocarcinogens that cause hepatocyte peroxisome proliferation, increased DNA synthesis, and decreased spontaneous apoptosis. We examined the effects of various PPs such as the hypolipidemic agents clofibric acid (CLO), bezafibrate (BEZA), ciprofibrate (CIPRO), and nafenopin (NAFE) and the plasticizer di-(2-ethylhexyl)phthalate (DEHP) on the various parameters in vitro in rat and human hepatocyte cultures. In rat hepatocyte cultures, after 72 h of treatment with the various PPs at 100–500 μM, a compound-dependent increase in acyl CoA oxidase (ACO) and carnitine acetyl transferase (CAT) activities, markers of peroxisome proliferation, was observed with the following potencies: CIPRO = NAFE > BEZA > CLO > DEHP. A minor (120–150%), but significant, no concentration-dependent increase in DNA synthesis and a marked, no compound-dependent and, with the exception of NAFE, no concentration-dependent 60–80% decrease in spontaneous apoptosis was observed with all tested compounds (50–250 μM) after 48 h of treatment. Inhibition of spontaneous apoptosis in PP-treated versus control rat hepatocyte cultures was also observed morphologically. Furthermore, PPs inhibited transforming growth factor β (TGFβ)-induced apoptosis but not tumor necrosis factor α (TNFα)/α Amanitine (αAma)-induced apoptosis in rat hepatocyte cultures. In human hepatocyte cultures, the various PPs at 50–500 μM did not affect peroxisomal enzyme activities, DNA synthesis, or spontaneous and induced (TGFβ or TNFα/αAma) apoptosis. The compound-dependent peroxisome proliferation but no compound-dependent disruption of the mitogenic/apoptotic balance elicited by PPs in primary rat hepatocyte cultures supports the hypothesis that oxidative stress is directly linked to the hepatocarcinogenic potential of a given PP in rodents and that disruption of the mitogenic/apoptotic balance contributes to the development of PP-induced hepatocarcinogenesis. In addition, the absence of effects of all PPs on both peroxisome proliferation-associated parameters and mitogenic/apoptotic balance supports the hypothesis that human liver cells are refractory to PP-induced hepatocarcinogenesis.

Chronic infection with hepatitis G virus in relation to hepatocellular carcinoma among non-Asians in Los Angeles County, California.

The recently identified hepatitis G virus (HGV) is a hepatotropic RNA virus belonging to the Flaviviridae family. The virus causes chronic viremia, and exposure to blood products is a recognized route of transmission in humans. To the authors' knowledge there is scant information regarding the hepatocarcinogenic potential of HGV. The current study examined the association between HGV infection and the risk of hepatocellular carcinoma. A population-based, case-control study involving 144 non-Asian patients with hepatocellular carcinoma who were ages 18-74 years at diagnosis and 252 community controls of similar age, gender, and race was conducted in Los Angeles, California. Study subjects were assessed for serologic markers of infections with the hepatitis B virus (hepatitis B surface antigen, antibody to the hepatitis B core antigen, and antibody to the hepatitis B surface antigen), hepatitis C virus (HCV) (anti-HCV and HCV RNA), and HGV (HGV RNA). Twelve of the 144 hepatocellular carcinoma patients (8.3%) and 5 of the 252 control subjects (2.0%) were positive for serum HGV RNA. The presence of HGV RNA in the serum was associated with a statistically significant 5.4-fold risk of hepatocellular carcinoma (95% confidence limit, 1.8, 16.6). The excess risk for hepatocellular carcinoma among HGV-infected individuals was independent of the effects of hepatitis B and hepatitis C infections. Chronic infection with HGV may play a role in the development of hepatocellular carcinoma. If the observed statistical association is a causal one, then infection with HGV may account for approximately 8% of hepatocellular carcinoma cases occurring in non-Asians in Los Angeles, California.

Suppression of hepatocyte apoptosis and induction of DNA synthesis by the rat and mouse hepatocarcinogen diethylhexylphlathate (DEHP) and the mouse hepatocarcinogen 1,4-dichlorobenzene (DCB).

Nongenotoxic rodent hepatocarcinogens do not damage DNA but cause liver tumours in the rat and mouse, associated with the induction of hepatic DNA synthesis. Previously, we have demonstrated that nongenotoxic hepatocarcinogens such as phenobarbitone and the peroxisome proliferator (PP), nafenopin, also suppress rat hepatocyte apoptosis. The nongenotoxic chemicals 1,4-dichlorobenzene (DCB) and the PP, diethylhexyl phthalate (DEHP), both induce high levels of DNA synthesis in rat liver in vivo, but only DEHP is hepatocarcinogenic in this species. Here, we investigate whether the difference in rat carcinogenicity of these two hepatic mitogens may be due to differences in their ability to suppress hepatocyte apoptosis. In rat hepatocytes in vitro, MEHP (the active metabolite of DEHP) induced DNA synthesis 2.5-fold (P = 0.001) and suppressed 10- and 4-fold, respectively both spontaneous (P = 0.0008) and transforming growth factor beta1 (TGFbeta1)-induced (P = 0.0001) apoptosis. DCB gave a small (1.7-fold) increase in DNA synthesis (P = 0.03) and a small (1.7- to 2-fold) suppression of both spontaneous (P = 0.022) and TGFbeta1-induced (P = 0.015) apoptosis. We next analysed the induction of DNA synthesis and the suppression of apoptosis in rat liver in vivo. Both DEHP and DCB were able to induce DNA synthesis although, as seen in vitro, the induction by DCB (4.2-fold; P = 0.023) was less marked than that with DEHP (13.4-fold; P = 0.007). Similarly, DEHP and DCB were both able to suppress rat hepatocyte apoptosis in vivo but the magnitude of the suppression was comparable; apoptosis was reduced to undetectable levels in four out of five animals with DCB and three out of five with DEHP. Since both chemicals suppressed apoptosis and induced DNA synthesis in rat liver but, overall, DCB was less potent, the disparate hepatocarcinogenic potential of these two chemicals could arise from differences in the magnitude of growth perturbation. To test this hypothesis, we repeated the studies in mouse, a species where both DCB and DEHP are hepatocarcinogenic. Both in vitro and in vivo, DCB and DEHP/MEHP were able to suppress apoptosis and induce hepatocyte DNA synthesis in the mouse with comparable potencies. The data support the hypothesis that the carcinogenicity of nongenotoxic hepatocarcinogens is associated strongly with the ability to perturb hepatocyte growth regulation. However, the ability to effect such changes is not unique to nongenotoxic carcinogens and is common to some noncarcinogenic chemicals, such as DCB, suggesting that the growth perturbation may need to exceed a threshold for carcinogenesis.

Does hepatitis GB virus-C infection cause hepatocellular carcinoma in black Africans?

The newly cloned and characterized hepatitis GB virus-C (HGBV-C), which is the same virus as the independently discovered hepatitis G virus, has a global distribution, is transmitted parenterally, and causes chronic viremia. The pathological consequences of infection with HGBV-C are uncertain, and its hepatocarcinogenic potential is unknown. We used a case-control format to compare the prevalence of HGBV-C infection in 167 southern African blacks with hepatocellular carcinoma (HCC) and 167 race-, age-, and sex-matched hospital-based control subjects, and to test for possible interactive effects between this virus and hepatitis B and C viruses in the development of the tumor. The presence of HGBV-C ribonucleic acid was detected in serum samples by reverse transcription, amplification of the resulting complementary deoxyribonucleic acid by the polymerase chain reaction (PCR), and Southern hybridization using a probe from the NS3/helicase region of the genome. Serum samples were also tested for the presence of hepatitis B virus surface antigen, antibodies to hepatitis C virus, and hepatitis C virus ribonucleic acid. Individuals infected with HGBV-C did not have an increased relative risk of developing HCC (relative risk 0.9; 95% confidence limits 0.5, 1.7). Moreover, co-infection with HGBV-C did not further increase the risk of tumor development in patients who were chronically infected with hepatitis B and/or C viruses. HGBV-C is unrelated to hepatocellular carcinoma development in black Africans.

Does hepatitis GB virus-C infection cause hepatocellular carcinoma in black Africans?

The newly cloned and characterized hepatitis GB virus-C (HGBV-C), which is the same virus as the independently discovered hepatitis G virus, has a global distribution, is transmitted parenterally, and causes chronic viremia. The pathological consequences of infection with HGBV-C are uncertain, and its hepatocarcinogenic potential is unknown. We used a case-control format to compare the prevalence of HGBV-C infection in 167 southern African blacks with hepatocellular carcinoma (HCC) and 167 race-, age-, and sex-matched hospital-based control subjects, and to test for possible interactive effects between this virus and hepatitis B and C viruses in the development of the tumor. The presence of HGBV-C ribonucleic acid was detected in serum samples by reverse transcription, amplification of the resulting complementary deoxyribonucleic acid by the polymerase chain reaction (PCR), and Southern hybridization using a probe from the NS3/helicase region of the genome. Serum samples were also tested for the presence of hepatitis B virus surface antigen, antibodies to hepatitis C virus, and hepatitis C virus ribonucleic acid. Individuals infected with HGBV-C did not have an increased relative risk of developing HCC (relative risk 0.9; 95% confidence limits 0.5, 1.7). Moreover, co-infection with HGBV-C did not further increase the risk of tumor development in patients who were chronically infected with hepatitis B and/or C viruses. HGBV-C is unrelated to hepatocellular carcinoma development in black Africans. (Hepatology 1997 Sep;26(3):740-2)

Hepatic Ploidy, Nuclearity, and Distribution of DNA Synthesis: A Comparison of Nongenotoxic Hepatocarcinogens with Noncarcinogenic Liver Mitogens

Most nongenotoxic hepatocarcinogens such as diethylhexyl phthalate (DEHP) and chlorendic acid (CEA) induce hepatocyte replicative DNA synthesis. However, not all mitogens are carcinogenic; in National Toxicology Program (NTP) studies 1,4-dichlorobenzene (DCB) was not hepatocarcinogenic in the rat despite the induction of substantial hepatic DNA synthesis. We have examined the hypothesis that the profile of hepatocyte labeling index (LI) may dictate hepatocarcinogenic potential. Using flow cytometry, we investigated the effects of DEHP, CEA, and DCB on hepatocyte ploidy, nuclearity, and LI distribution among the ploidy/nuclearity classes in male Fischer 344 rats. Dosing was for 7 days at the dose and route employed previously in the NTP cancer bioassays. Unlike DEHP and CEA, DCB reduced the proportion of tetraploid cells (4N plus 2 × 2N) and increased the proportion of mononucleated octoploid (8N) cells. DCB and DEHP increased the mean hepatic LI (17 ± 2 and 23 ± 3%, respectively, compared with 1.4 ± 0.4% in controls), whereas, as expected for a chronic inducer of S-phase, CEA did not. LI was increased in all hepatocyte ploidy/nuclearity classes except the binucleated tetraploid cells (2 × 2N) and was elevated most in the mononucleated octoploid population (8N) (LI = 44 ± 11, 49 ± 14, and 1.3 ± 0.4% of 8N hepatocytes for DCB, DEHP, and control, respectively). In general, the effects of DCB on LI could not be distinguished from those of DEHP. However, DEHP tended to induce DNA synthesis in a greater proportion of 2N and 2 × 4N cells; future studies will analyze whether the induction of DNA synthesis in these small populations may be more relevant to hepatocarcinogenesis.

Expression of the Immediate-Early Genes, c-fos, c-jun, and c-myc: A Comparison in Rats of Nongenotoxic Hepatocarcinogens with Noncarcinogenic Liver Mitogens

The involvement of the immediate-early (IE) genes c-fos, c-jun, and c-myc in regenerative liver hyperplasia is accepted, but their involvement in direct hyperplasia is uncertain. We have examined the hypothesis that the ability to induce IE genes may reflect the hepatocarcinogenic potential of a chemical. The ability of 1,4-dichlorobenzene (DCB) (300 mg/kg) (a noncarcinogemc rat liver mitogen), diethylhexyl phthalate (DEHP) (950 mg/kg), and chlorendic acid (120 mg/kg) (both nongenotoxic hepatocarcinogens) to induce c-fos, c-jun. and c-myc expression in rat liver was determined by Northern blot analysis and by in situ hybridization. Results were correlated to hepatic labeling index (LI) as determined by incorporation of BrdU in each of three lobes for each of three male F344 rats per group. Carbon tetrachioride (CCl4 (2 ml/kg) was used as a positive control. Increased LI was preceded by elevated expression of all three IE genes after CCl4., but also after DCB and DEHP, although induction by these was less marked. In all cases, there was considerable interanimal variation within groups, but little interlobe variation. Interestingly, there was a good correlation (r2 ≥ 0.85) between c-myc expression and LI, but not between LI and c-fos or c-jun. Despite the disparate carcinogenic potential of DEHP and DCB, both chemicals induced similar patterns of IE gene expression, suggesting that this cannot distinguish hepatocarcinogenic liver mitogens from noncarcinogenic liver mitogens. These data assist in the evaluation of IE gene expression both as a marker of direct versus regenerative hyperplasia and as an indicator of the hepatocarcinogenic potential of liver mitogens.