Prevention of Lipid Peroxidation-derived Cyclic DNA Adduct and Mutation in High-Fat Diet-induced Hepatocarcinogenesis by Theaphenon E.

Obesity is associated with cancer risk and its link with liver cancer is particularly strong. Obesity causes non-alcoholic fatty liver disease (NAFLD) that could progress to hepatocellular carcinoma (HCC). Chronic inflammation likely plays a key role. We carried out a bioassay in the high-fat diet (HFD)-fed C57BL/6J mice to provide insight into the mechanisms of obesity-related HCC by studying γ-OHPdG, a mutagenic DNA adduct derived from lipid peroxidation. In an 80-week bioassay, mice received a low-fat diet (LFD), high-fat diet (HFD), and HFD with 2% Theaphenon E (TE) (HFD+TE). HFD mice developed a 42% incidence of HCC and LFD mice a 16%. Remarkably, TE, a standardized green tea extract formulation, completely blocked HCC in HFD mice with a 0% incidence. γ-OHPdG measured in the hepatic DNA of mice fed HFD and HFD+TE showed its levels increased during the early stages of NAFLD in HFD mice and the increases were significantly suppressed by TE, correlating with the tumor data. Whole-exome sequencing showed an increased mutation load in the liver tumors of HFD mice with G>A and G>T as the predominant mutations, consistent with the report that γ-OHPdG induces G>A and G>T. Furthermore, the mutation loads were significantly reduced in HFD+TE mice, particularly G>T, the most common mutation in human HCC. These results demonstrate in a relevant model of obesity-induced HCC that γ-OHPdG formation during fatty liver disease may be an initiating event for accumulated mutations that leads to HCC and this process can be effectively inhibited by TE. Cancer Prev Res; 11(10); 665-76. ©2018 AACR.

Obesity, but not ethanol, promotes tumor incidence and progression in a mouse model of hepatocellular carcinoma in vivo.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major global health burden. Although chronic, heavy alcohol abuse is an established risk factor for HCC, obesity is emerging as an increasingly important factor in HCC development. Given that other risk factors for HCC act synergistically to promote tumorigenesis, we investigated the effects of diet-induced obesity and chronic ethanol consumption on tumor progression. METHODS: A diethylnitrosamine (DEN) mouse model of HCC was established and mice randomized to control (CD; 10 % kcal% fat) or high fat (HFD; 60 % kcal% fat diet) at 5 weeks of age. At 35 weeks, mice were randomized to 10/20 % ethanol (EtOH) in drinking water (alternate days), or drinking water (H2O) alone. Tumor incidence/size were measured and confirmed. Liver tissue was analyzed for oxidative stress and EtOH-metabolizing enzymes and serum analyzed for liver function and nutritional status. RESULTS: DEN treatment induced HCC formation in 60 % CD-H2O mice (6 of 10), an effect exacerbated by HFD (89 %). Tumors in HFD animals occupied significantly more of the liver than mice on CD. EtOH-feeding did not impact HCC incidence or tumor size. HFD resulted in increased liver injury and liver:body weight ratio regardless of EtOH consumption. Increased tumor incidence was associated with elevated hepatic oxidative stress in the absence of changes in intrinsic antioxidant (glutathione) levels. CONCLUSIONS: Obesity independently promoted HCC formation in the absence or presence of a known hepatocarcinogen (DEN), and enhanced both number and size of hepatic tumors independent of chronic EtOH consumption in mice.

Cytokines as important playmakers of experimental hepatocarcinogenesis confounded by diabetes.

PURPOSE: To explore the possible intermediary pathways through which diabetes mellitus (DM) adversely worsens hepatocellular carcinoma (HCC), focusing on cell life controllers as some transcription factors and inflammatory mediators. MATERIAL AND METHODS: Forty male albino rats were divided into four groups, control, cancer [given single intra-peritoneal (IP) dose of diethyl nitrosamine, NDEA, 125 mg/kg body weight], diabetic (given single dose of streptozotocin, STZ, 65 mg/kg) and cancer diabetic. HCC was initiated with NDEA, 3 weeks later, DM was induced with STZ. At 14th week, animals were sacrificed. Serum ALT, AST, GGT activities, AFP, IL-6, TNF-α levels and liver tissue Bax and Bcl2 proteins were measured. Liver sections were stained for histological examination. Both histological and AFP variations were chosen to prove cancer development. RESULTS: NDEA group showed significant increase in liver weight, serum ALT, AST, GGT, AFP, TNF-α, IL-6 and liver Bcl2 protein with decrease in total body weight, liver Bax protein and Bax/Bcl2 ratio. These effects were more pronounced in DENA plus STZ group. IL-6, TNF-α and Bcl2 were positively correlated while Bax and Bax/Bcl2 ratio were negatively correlated to AFP levels reflecting potential diagnostic value. CONCLUSION: Co-induction of DM in the course of hepatocarcinogenesis can dramatically influence disease progression through inflammation and retarded apoptosis. The suggested apoptotic and inflammatory markers seem to be beneficial diagnostic tools for HCC and improve the diagnostic performance of AFP.

The effect of dietary restriction on PhIP-induced mutation in the distal colon and B[a]P- and ENU-induced mutation in the liver of the rat.

A reduction in dietary intake has been shown to significantly increase the lifespan of rodents, lower the incidence of tumors, and reduce DNA damage. The objective of this study was to determine whether dietary restriction (DR) reduced the frequency of mutation induced by two environmentally relevant metabolically activated mutagens and one direct-acting mutagen in the lacI transgene of male and female Big BlueR rats. Both male and female rats were maintained on either an ad libitum (AL) or a 40%-reduced diet for 22 wk. The mutagenicity of a 100-mg/kg intraperitoneal injection with 2-amino-1-methyl-6-pheny-imidazo[4,5-b] pyridine (PhIP), benzo[a]pyrene (B[a]P), and N-ethyl-N- nitrosourea (ENU) was determined in the colon or liver. The results indicated that DR did not significantly alter the PhIP-induced mutant frequency in male or female colons. DR completely prevented mutagenicity induced by B[a]P in the female liver (2.6 +/- 0.6 10(-5) vs 10.9 +/- 5.8 10(-5) in AL females), yet increased the induced frequency in male livers (16.3 +/- 3.7 10(-5) vs 10.6 +/- 1.5 10(-5) in AL male livers). Although there was no difference in mutation frequency in the liver between AL and DR females treated with ENU, there was approximately a 40% decrease in induced frequency in DR males compared with AL males. These results indicate that a reduction in dietary intake has no preventive effect against PhIP-induced mutation in the colon, but has sex-dependent protective effects against B[a]P- and ENU-induced mutation in the liver.

Liver carcinogenesis and formation of 8-hydroxy-deoxyguanosine in C3H/HeN mice by oxidized dietary oils containing carcinogenic dicarbonyl compounds.

Oxidized dietary oils (lard, soybean oil, and sardine oil) were orally administered to C3H/HeN male mice. After 6 months, benign hepatocellular adenoma was observed in the mice treated with all three oxidized dietary oils. After 12 months, malignant hepatocellular carcinoma and hepatoblastoma were observed in addition to the benign tumor. Oxidized sardine oil caused the highest tumor incidence (35%) and malignant tumors (27.5%) among the oxidized dietary oils tested. Mice treated with oxidized lard and sardine oil exhibited a significant increase of 8-OH-dG in the livers. The amounts of 8-OH-dG found in the mice treated with oxidized sardine oil correlated with the rates of tumor incidence. After 6 months, mRNA decreased in the case of oxidized lard and sardine oil, whereas it increased in the case of oxidized soybean oil, either in 8-oxoguanine-DNA glycosylase (OGG1) or in 8-oxo-dGTPase. On the other hand, there was no appreciable change in mRNA, in either OGG1 or 8-oxo-dGTPase, after 12 months. Oxidized sardine oil contained the highest level of malonaldehyde (MA) (713+/-91.1 nmol/g) and glyoxal (33.3+/-5.2 nmol/g) among three oxidized oils. The malignant tumor incidence correlated with the high level of MA and glyoxal found in the dietary oils tested.

Suppression of altered hepatic foci development by curcumin in wistar rats.

Curcumin, a yellow pigment of turmeric (Curcuma longa), is a commonly used spice and a coloring agent in foods, drugs, and cosmetics. Curcumin is known to possess chemopreventive properties in various animal tumor models. In the present study the effect of curcumin on the development of altered hepatic foci (AHF), by using a medium term liver bioassay, has been evaluated. AHF were analyzed by quantitative stereology using the Leica Qwin Image Analysis system from frozen liver sections stained for g-glutamyl transferase, adenosine triphosphatase, glucose-6-phosphatase, alkaline phosphatase, and placental isozyme of glutathione S-transferase. A significant protection on diethylnitrosamine (DEN) initiated and 2-acetylaminofluorene (AAF) promoted AHF by curcumin was observed on these biological markers. The curcumin administration was found to restore the normal levels of the enzymes glutathione S-transferase and g-glutamyl transferase in rat liver following DEN-AAF exposure. Similarly, a significant protection was provided by curcumin in the enzyme-deficient foci for the adenosine triphosphatase-, alkaline phosphatase-, and glucose-6-phosphatase-treated groups in comparison to the DEN-AAF-treated group. These results show that curcumin can effectively suppress the DEN-induced development of AHF in rat liver.

A Wistar rat strain prone to spontaneous liver tumor development: implications for carcinogenic risk assessment.

The European Union legislation considers nongenotoxic substances that only cause liver tumors in certain sensitive strains of mice as raising no concern for man. The EU legislation, however, also clearly stipulates that cases where the only available tumor data are the occurrence of neoplasms at sites and in strains where they are well known to occur spontaneously with a high incidence are relevant arguments which exclude a concern for man. We have analyzed the spontaneous liver tumor incidence in Wistar rats and in B6C3F(1) and C57Bl mice used in carcinogenicity trials at the BASF facility in Ludwigshafen, Germany, over the past 15 years and compared the spontaneous liver tumor incidence in BASF Wistar rats with those observed in rat strains employed in major European contract research organizations (CROs). The results of these analyses indicate that the incidence of spontaneous liver tumors in the BASF Wistar rat strain is very high, similar to that seen in the B6C3F(1) mouse and much higher than that seen in the C57Bl mouse and other commonly used strains of rat. The analyses also revealed signs of increasing variability and liver tumor drift in several rat strains. Moreover, the incidence of spontaneous preneoplastic liver cell foci was far higher in the BASF Wistar rat strain than reported for other rat strains in the literature. The analyses provide relevant arguments which exclude a concern for man for nongenotoxic chemicals that only tested positive for liver tumors in this sensitive rat strain.

Further evidence for chemopreventive potential of beta-carotene against experimental carcinogenesis: diethylnitrosamine-initiated and phenobarbital-promoted hepatocarcinogenesis is prevented more effectively by beta-carotene than by retinoic acid.

The comparative effectiveness of beta-carotene (BC) and retinoic acid (RA) was investigated against two-stage rat liver carcinogenesis initiated by a single injection of diethylnitrosamine (DEN, 200 mg/kg i.p.) followed by promotion with phenobarbital (PB, 0.05%) in a basal diet. BC (500 mg/kg) or RA (200 mg/kg) was administered per os daily throughout the entire experiment, before the initiation, or during the promotional stage. Treatment with BC throughout the experiment or before initiation significantly reduced the incidence (p < 0.01), multiplicity (p < 0.05), and size of visible subcapsular hepatocyte nodules (HNs) and reduced (p < 0.001 or 0.05) nodular volume as a percentage of liver volume. The results with RA were of lesser extent than those observed with BC. There was a considerable depletion of hepatic BC and total vitamin A (retinol + ester) in HNs and nonnodular surrounding parenchyma (NNSP) of rats subjected to the DEN-PB regimen than their control counterparts. Treatment with BC significantly elevated hepatic BC and total vitamin A contents in HNs and NNSP compared with DEN-PB control, and the elevation was proportional to the duration of BC treatment. Long-term BC or RA treatment elicited a substantial decrement in reduced glutathione content and gamma-glutamyltranspeptidase activity and an increment in cytochrome P-450 content and glutathione peroxidase and glutathione S-transferase activities in the HNs and NNSP, which were otherwise reversed in rats that received DEN-PB treatment alone. Our results suggest that BC or RA has the potential to inhibit DEN-induced hepatocarcinogenesis through selective modulation of the antioxidant defense system and xenobiotic detoxification in the liver. It is also apparent that the beneficial effect of BC or RA is primarily exerted on the initiation phase and secondarily during the promotional stage of DEN-initiated rat liver carcinogenesis and that BC affords a better chemopreventive response than RA.

Histopathology of Japanese medaka (Oryzias latipes) chronically exposed to a complex environmental mixture.

Japanese medaka (Oryzias latipes) were used to evaluate the carcinogenicity of a complex groundwater that contained 5 U.S. Environmental Protection Agency priority pollutant heavy metals and 13 chlorinated aliphatic hydrocarbons. A test protocol that used 10 mg/L diethylnitrosamine (DEN) prior to groundwater exposure was designed to assess both initiation and promotion. The fish were exposed continuously for 9 mo with 0, 1, 5, or 25% groundwater, by volume, with either West Branch of Canal Creek water (Aberdeen Proving Ground-Edgewood Area, Aberdeen Proving Ground, MD) or dechlorinated tap water as the diluent, while concurrent controls were run in the laboratory. Incidental findings included various neoplasms in the nares, ovary, skeletal muscle, skin, swim bladder, testis, thymus, and thyroid. Factors evaluated during statistical analyses of fish neoplasm prevalence included diluent type, groundwater percentage, fish gender, and DEN initiation. Liver neoplasm prevalence was higher in DEN-initiated fish and was frequently higher in males. Concentrations of up to 25% groundwater, by volume, showed no evidence of being a complete carcinogen and showed no consistent, conclusive evidence of being a promoter.

Effect of fasting/refeeding on the incidence of chemically induced hepatocellular carcinoma in the rat.

Caloric restriction has been associated with a delay in the development of both spontaneous and induced neoplasia. In contrast, cycles of fasting/refeeding were shown by us and others to enhance the incidence of early lesions during chemical carcinogenesis in rat liver. The present, long-term study was undertaken to establish whether such a diffential effect would also extend to the later phases of cancer development, until the overt appearance of neoplasia. Male Fischer 344 rats were initiated with a single dose of diethylnitrosamine (DENA, 200 mg/kg i.p.) and starting 1 week later they were either exposed to three cycles of fasting (3 days) followed by refeeding (11 days) or were fed continuously. Seven weeks after DENA administration the rats were exposed to the resistant hepatocyte model of the liver tumor promotion protocol. All animals were killed 1 year after initiation. Incidence of hepatocellular carcinoma was 2-fold higher in the fasted/refed group compared with the controls (72 versus 36%). In addition, cancers were also larger and of higher histological grade in the former group, with one animal showing metastases to the lungs, while no metastases developed in control animals. Fasting caused a decrease in total liver DNA (from 25.2 +/- 1.1 to 16.5 +/- 1.1 mg after 3 days) which was associated with a decrease in hepatocyte labeling index and mitotic activity and high levels of single cell death (apoptosis). In contrast, a sharp increase in hepatocyte proliferation was observed on day 2 of refeeding and this was more pronounced in glutathione S-transferase 7-7 positive foci compared with surrounding liver (10.2 +/- 2.3 versus 4.6 +/- 0.8%). Such a proliferative wave was associated with a sharp decline in the incidence of cell death. It is concluded that fasting/refeeding performed early after initiation accelerates the development of chemically induced hepatocellular carcinoma in the rat.

The tamoxifen dilemma.

The anti-oestrogen tamoxifen is widely used for adjuvant therapy in the treatment of women with breast cancer and has a low incidence of serious side-effects. It could also play a role as a breast cancer chemopreventive agent. However, epidemiological studies in both tamoxifen-treated breast cancer patients and in healthy women have shown that treatment results in a small increase in the incidence of endometrial cancers. While the use of tamoxifen in breast cancer patients is clearly justified, the situation for its use as a chemopreventive agent in healthy women is not so clear cut. Reasons for caution come from studies in rats that show that tamoxifen is a genotoxic mutagenic liver carcinogen. Initiation of tumours in the rat is the result of metabolic activation of tamoxifen by CYP enzymes to an electrophile(s) that binds irreversibly to DNA. This is not related to the oestrogen receptor status of the tissue. The extent of DNA damage, detected by 32P-post-labelling or accelerator mass spectrometry, is dependent both on the dose and the length of exposure. Studies have been carried out to see if such binding occurs in the uterine endometrium from tamoxifen-treated women. Results are presently inconclusive, but if such irreversible DNA binding occurs, it is at very low levels. Based on a mechanistic understanding of tamoxifen-induced liver carcinogenesis in the rat, it seems that in humans hepatic DNA damage will be close to the limit of detection by 32P-post-labelling and liver cancer will not be a significant carcinogenic risk. We cannot be certain of the mode of action of tamoxifen that results in the increase in endometrial cancers in treated women but it seems unlikely that this will be associated with a classical genotoxic mechanism.

Trichloroethylene cancer risk: simplified calculation of PBPK-based MCLs for cytotoxic end points.

Cancer risk assessments for trichloroethylene (TCE) based on linear extrapolation from bioassay results are questionable in light of new data on TCE's likely mechanism of action involving induced cytotoxicity, for which a threshold-type dose-response model may be more appropriate. Previous studies have shown that if a genotoxic mechanism for TCE is assumed, algebraic methods can considerably simplify the use of physiologically based pharmacokinetic (PBPK) models to estimate virtually safe environmental concentrations for humans based on rodent cancer-bioassay data. We show here how such methods can be extended to the case in which TCE is assumed to induce cancer via cytotoxicity, to estimate environmentally safe concentrations based on rodent toxicity data. These methods can be substituted for the numerical methods typically used to calculate PBPK-effective doses when these are defined as peak concentrations. We selected liver and kidney as plausible target tissues, based on an analysis of rodent TCE-bioassay data and on a review of related data bearing on mechanism. Tumor patterns in rodent bioassays are shown to be consistent with our estimates of PBPK-based, effective cytotoxic doses to mice and rats used in these studies. When used with a margin of exposure of 1000, our method yielded maximum concentration levels for TCE of 16 ppb (87 micrograms/m3) for TCE in air respired 24 hr/day, 700 ppb (3.8 mg/m3) for TCE in air respired for relatively brief daily periods (e.g., 0.5 hr while showering/bathing), and 210 micrograms/liter for TCE in drinking water assuming a daily 2-liter ingestion. Cytotoxic effective doses were also estimated for occupational respiratory exposures. These estimates indicate that the current OSHA permissible exposure limit for TCE would produce metabolite concentrations that exceed an acute no observed adverse effect level for hepatotoxicity in mice. On this basis, the OSHA TCE limit is not expected to be protective.

Enhancement of hepatocarcinogenesis by combined administration of food-derived heterocyclic amines at low doses in the rat.

Effects of simultaneous administration of five or 10 heterocyclic amines (HCAs) at low dose levels on rat liver carcinogenesis were investigated using a medium-term bioassay protocol. Male F344 rats were initially given diethylnitrosamine (DEN, 200 mg/kg, ip) and received HCAs starting 2 wk later for 6 wk. All animals were subjected to two-third partial hepatectomy at wk 3 and were killed at wk 8. Five carcinogenic HCAs in the first two experiments: 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 2-aminodipyrido[1,2-alpha: 3',2'-d]imidazole, 2-amino-3-methylimidazo-[4,5-f]quinoxaline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline in experiment 1 and 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-6-methyl-dipyrido[1,2-alpha: 3',2'-d]imidazole,2-amino-3-methyl- 9H-pyrido-[2,3-b]indole, 2-amino-9H-pyrido[2,3-b]indole, and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine in experiment 2] were administered together or individually in the diet at levels of 1/1, 1/5 or 1/25 carcinogenic doses, and all 10 chemicals were given at 1/10 or 1/100 levels in experiment 3. Induction of preneoplastic glutathione S-transferase placental from (GST-P) positive foci was increased in some combination groups over the sums of effects for the individual groups at the same doses (apparent synergism). This was most obvious in the group given all 10 chemicals at the 1/10 dose levels. However, it was of interest that the values in the combined groups were generally very close to the averages of five or 10 individual results for the corresponding higher dose groups, indicating isoadditivity of HCA effects. True (strict) synergism, however, was expected for the results of groups including PhlP and Trp-P-2 in combination, since they are non-hepatocarcinogenic but induce the key metabolic enzyme for HCAs (CYP1A2).

Effects of pesticide mixtures at the acceptable daily intake levels on rat carcinogenesis.

Possible modifying effects of pesticide mixtures on tumorigenesis were investigated with medium-term carcinogenesis protocols for rapid detection of carcinogenic agents using male F344 rats. In the 8-wk liver model, administration of 20 pesticides (19 organophosphorus compounds and one organochlorine), added to the diet each at acceptable daily intake (ADI) levels, did not enhance rat liver preneoplastic lesion development initiated by diethylnitrosamine. In contrast, a mixture of these 20 pesticides at 100 times the ADI significantly increased the number and area of liver lesions. In the second experiment using a multi-organ carcinogenicity protocol of 28 wk, mixtures of 40 pesticides (high production examples) or 20 pesticides (suspected carcinogens) added to the diet at their respective ADI levels did not modulate carcinogenesis in any organ initiated by five known potent carcinogens in combination. These results thus provide direct support for the safety factor (usually 100) approach using ADI values for the quantitative risk evaluation of pesticides.

Dose-dependent induction of GST-P+ staining foci by the rat hepatocarcinogen methapyrilene in the medium-term bioassay.

Previous studies have demonstrated that methapyrilene hydrochloride (MP) is a rat-specific nongenotoxic carcinogen which induces liver tumors in a dose-dependent manner following chronic exposure in the diet. This study was conducted to determine the dose response of MP in the medium-term bioassay and to compare the response to tumor incidence. Two weeks following a single initiating dose of diethylnitrosamine (DEN), male F344 rats were administered MP at doses of 0, 62.5, 125, 250, or 1000 ppm in the diet for 6 weeks. A 2/3 partial hepatectomy was performed 3 weeks post-DEN. At termination, sections from the remaining three lobes were stained with GST-P antibody. Number and size of foci were measured using an image analysis system with a digitizing board. MP induced a dose-dependent increase in the number of GST-P+ foci/cm2 (0 ppm = 0.85 foci/cm2; 62.5 ppm = 1.29 foci/cm2; 125 ppm = 1.59 foci/cm2; 250 ppm = 6.55 foci/cm2; 1000 ppm = 28.23 foci/cm2). A significantly greater number of foci were observed in the caudate lobe than in the anterior and posterior lobes. The size of individual foci was largely unaffected. This study demonstrates a strong correlation between foci induction and tumor incidence and suggests that this assay may have utility in predicting dose responses for the chronic bioassay.

The effect of body weight on tumor incidence and carcinogenicity testing in B6C3F1 mice and F344 rats.

Associations between animal body weights and tumor incidence were examined using individual control animal data from 55 mouse and 53 rat studies conducted by the National Toxicology Program. Several statistically significant associations were found, the strongest of which were positive relationships between body weight and risk of liver tumors in both sexes of mice, pituitary gland tumors in both sexes of rats, and mammary gland tumors in female rats. The most compelling evidence that these relationships were causal in nature was the replication of the correlations across individual experiments. In addition, significant correlations between tumor occurrence and body weights occurred in animals as young as 9 weeks of age. Logistic regression models relating 12-month body weight to tumor risk were developed for the strongest relationships, and utilized in the reanalysis of tumor data from two National Toxicology Program studies with treatment-related decreases in body weight. A simulation study based on the logistic regression models indicated that statistical power to detect a treatment-related increase in tumor incidence can be seriously diminished when mean body weight in treated animals is depressed by as little as 10%. Conversely, when mean body weight in control animals is 10% lower than that of treated animals, false positive rates can increase to 20-30%. The severity of the effects of such commonly observed treatment-related disparities in body weight suggests that alternative data analysis methods or experimental designs should be considered to address this potential problem in carcinogenicity testing.

Dissimilar frequency of hepatoblastomas and hepatic cystadenomas and adenocarcinomas arising in hepatocellular neoplasms of D2B6F1 mice initiated with N-nitrosodiethylamine and subsequently given Aroclor-1254, dichlorodiphenyltrichloroethane, or phenobarbital.

Aroclor-1254 (Ar-1254) and dichlorodiphenyltrichloroethane (DDT) were compared to phenobarbital (PB) for their ability to promote hepatocellular proliferative lesions to hepatocellular adenomas and carcinomas and to hepatoblastomas in D2B6F1 male mice initiated with N-nitrosodiethylamine (NDEA). Hepatocellular neoplasms developed in all mice given NDEA and were more numerous in mice fed promoters. Multiplicities decreased in the order Ar-1254 > PB > DDT, indicating that Ar-1254 was more potent than either PB or DDT at the dosage levels used. PB was the most effective of the 3 agents in stimulating the evolution of hepatocellular neoplasms to hepatoblastoma. The incidence of hepatoblastomas in the NDEA.PB group was 72% but was only 27% in NDEA-initiated, DDT-promoted mice and 33% in low-dose and only 9% in high-dose Ar-1254-promoted mice. In contrast, lesions resembling benign and malignant cholangiocellular neoplasms were frequently found within hepatocellular tumors in Ar-1254-promoted mice but not in mice fed PB or DDT, either alone or after NDEA. Some cystic glandular structures in Ar-1254-promoted mice contained mucous cells, argentaffin cells, and Paneth cells and thus constituted intestinal metaplasia. Hepatoblastoma and intestinal metaplasia/cholangiocellular tumor morphology appear to constitute different patterns of genetic programming induced by certain promoters in expanding clones of initiated hepatocytes, on favorable genetic backgrounds such as that of D2B6F1 male mice.

Induction of lung and liver tumors by fluoranthene in a preweanling CD-1 mouse bioassay.

Fluoranthene (FA), a major environmental pollutant, induced lung and liver tumors 6-9 months after intraperitoneal injection of 0.7, 1.75 and 3.5 mg FA into preweanling CD-1 mice. There was a dose-dependent increase in lung tumors with a maximum tumor incidence of nearly 45% and a maximum tumor multiplicity of 0.6-0.7 lung tumors/mouse. No significant difference was noted in lung tumors in the 6 and 9 month bioassays, although fewer tumors were consistently noted in mice treated with the two lowest doses of FA. Indices of lung tumor incidence (ED50) and multiplicity (TM1.0) were similar for the two bioassays and ranged from 18.9-19.5 and 26.2-27.2 mumol respectively. Male mice had up to two times more lung tumors than females but these results were not statistically significant. Liver tumors (nodular hyperplasia) appeared only in FA-treated males but no dose-response relationship was evident. However, liver tumors were observed in only 0-10% of the male mice in the 6 month treatment groups, but in 20-60% of the males in the 9 month groups. Because the CD-1 preweanling mouse responded to the weak lung tumorigen FA, it is a viable, limited-term bioassay for measuring tumorigenicity of PAH and combustion emissions.

Thyroid follicular cell carcinogenesis: results from 343 2-year carcinogenicity studies conducted by the NCI/NTP.

The National Toxicology Program data base on 343 mouse and rat carcinogenesis studies was reviewed to determine the frequency of and relationship between hyperplastic and neoplastic follicular lesions of the thyroid gland. The frequency of chemically related lesions in the thyroid was also compared to neoplastic lesions in the liver to investigate a possible correlation. The percentage of studies observed to have positive or equivocal chemically related thyroid proliferative lesions was rats: male, 14%, female, 11%; mice; male, 8%; female, 9%. When positive in one sex for a given chemical, there was a 60-80% chance of it being positive in the other sex of the same species, although interspecies correlation was not as strong. Thyroid follicular cell neoplasia without hyperplasia was uncommon in mice but was common in rats. Chemicals that caused thyroid proliferative changes were more likely (P less than 0.05) to produce liver neoplasms (both within and between species) than were chemicals causing no thyroid changes. However, this correlation was far from perfect, with many chemicals producing thyroid proliferative lesions, but not liver neoplasms and vice versa. This suggests that universal correlations are not supportable by the data and that individual chemicals should be evaluated on a case-by-case basis.

Promoting effect of ovariectomy on hepatocellular tumorigenesis induced in mice by 3'-methyl-4-dimethylaminoazobenzene.

The treatment of female C57BL/6 x DS-F1 mice with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) at 10, 12, 14, 16 and 18 days of age resulted in the development of hepatocellular adenomatous nodules after 10 months of age. Ovariectomy in these mice at 1 month of age hastened the development of adenomatous nodules, which then first appeared at 8 months of age. The incidence of adenomatous nodules in females ovariectomized at the age of 1 month was much higher than that in intact females of the same age. These results showed that the ovaries exerted a suppressive effect on the development of adenomatous nodules. To determine the time from which the ovaries exert this suppressive effect, females were ovariectomized at 4, 6, 8, and 10 months of age, and the incidences of adenomatous nodules were compared at 10 and 12 months of age. Delayed ovariectomy after 8 months of age did decrease the incidence of adenomatous nodules at 10 and 12 months of age, but ovariectomy after 4 and 6 months of age did not. When the incidence of adenomatous nodules in females ovariectomized at 10 months of age was examined over the subsequent 6 months, it became significantly higher after 14 months of age compared with that in intact females. The results show that the ovariectomy has the promoting effect on the development of adenomatous nodules in the liver induced by 3'-Me-DAB after 6 months of age.