Furan-induced dose-response relationships for liver cytotoxicity, cell proliferation, and tumorigenicity (furan-induced liver tumorigenicity).

Rodent studies of furan are associated with liver cell necrosis, release of liver-associated enzymes, increased hepatocyte proliferation, and hepatocarcinogenesis. For carcinogens whose proposed mode of action is cytolethality, it is hypothesized that the dose-response curve for tumor development would parallel the dose-response curve for cell death with compensatory proliferation in the target organ. To prospectively test this hypothesis, female B6C3F(1) mice were exposed to furan at carcinogenic doses and lower for 3 weeks or 2 years. At 3 weeks and in the 2-year study, there were dose-dependent and significant increases in hepatic cytotoxicity at 1.0, 2.0, 4.0, and 8.0mg furan/kg. For cell proliferation as measured by 5-bromo-2'-deoxyuridine (BrdU) labeling index (LI), there was a statistically significant trend with increasing dose levels of furan and increased LI at 8.0mg/kg. There was an increased incidence of foci of altered hepatocytes, hepatocellular adenomas, and adenomas or carcinomas at 4.0 and 8.0mg/kg and carcinomas at 8.0mg/kg. The multiplicity of microscopic tumors was increased and latency was decreased in mice exposed to 8.0mg/kg. Prevalence of hepatic nodules at necropsy was increased in mice exposed to 4.0 and 8.0mg/kg. Data demonstrate an association among furan-induced hepatic cytotoxicity, compensatory cell replication, and liver tumor formation in mice; at high doses >or=4.0mg/kg, furan induced hepatotoxicity, compensatory cell replication and tumorigenesis in a dose-related manner, while furan did not produce tumors at cytotoxic doses of 1.0 and 2.0mg/kg.

Kinetics of arsenic methylation by freshly isolated B6C3F1 mouse hepatocytes.

The toxic and carcinogenic effects of arsenic may be mediated by both inorganic and methylated arsenic species. The methylation of arsenic(III) is thought to take place via sequential oxidative methylation and reduction steps to form monomethylarsenic (MMA) and dimethylarsenic (DMA) species, but recent evidence indicates that glutathione complexes of arsenic(III) can be methylated without oxidation. The kinetics of arsenic methylation were determined in freshly isolated hepatocytes from male B6C3F1 mice. Hepatocytes (>90% viability) were isolated by collagenase perfusion and suspended in Williams' Medium E with various concentrations of arsenic(III) (sodium m-arsenite). Aliquots of the lysed cell suspension were analyzed for arsenic species by hydride generation-atomic absorption spectrometry. The formation of MMA(III) from sodium arsenite (1 microM) was linear with respect to time for >90 min. DMA(III) formation did not become significant until 60 min. MMA(V) and DMA(V) were not consistently observed in the incubations. These results suggest that the glutathione complex mechanism of methylation plays an important role in arsenic biotransformation in mouse hepatocytes. Metabolism of arsenic(V) was not observed in mouse hepatocytes, consistent with inhibition of arsenic(V) active cellular uptake by phosphate in the medium. The formation of MMA(III) increased with increasing arsenic(III) concentrations up to approximately 2 microM and declined thereafter. The concentration dependence is consistent with a saturable methylation reaction accompanied by uncompetitive substrate inhibition of the reaction by arsenic(III). Kinetic analysis of the data suggested an apparent K(M) of approximately 3.6 microM arsenic(III), an apparent V(max) of approximately 38.9 microg MMA(III) formed/L/h/million cells, and an apparent K(I) of approximately 1.3 microM arsenic(III). The results of this study can be used in the physiologically based pharmacokinetic model for arsenic disposition in mice to predict the concentration of MMA(III) in liver and other tissues.

An investigation of the effects of late-onset dietary restriction on prostate cancer development in the TRAMP mouse.

In our previous work we showed that dietary restriction initiated at puberty reduced prostate cancer development in the TRAMP mouse model. The current study was conducted to ascertain whether a dietary restriction regime would similarly reduce lesion development if imposed once tumor development was well established. Male TRAMP mice were maintained on an ad libitum diet until 20 weeks of age when proliferative prostate lesions are clearly evident. Mice were then subjected to a 20% restriction in dietary calories compared to matched controls, which were continued on ad libitum feeding. Mice were sacrificed at 20, 24, 32, and 39 weeks of age and proliferative epithelial lesions of the prostate were assessed using an established grading scheme. In this study, although dietary restriction reduced mean sex pluck weight (prostate and seminal vesicles), and mean grade of epithelial proliferative lesions in the dorsal and lateral lobes of the prostate, the effect was not as pronounced as was the case with dietary restriction from puberty. There was no relationship between serum insulin like growth factor (IGF-1) and prostate lesion grade. Additionally, we also report the relationship between lobe specific lesion development and SV40 immunostaining and, the occurance of neuroendocrine tumors (NETs) in the ventral prostate and urethra of the TRAMP mouse. NETs stained with high specificity and sensitivity for the neuroendocrine markers, synaptophysin and neuron-specific enolase (NSE), less for serotonin, but not for chromogranin A. NETs did not stain for cyclo-oxygenase-2 (COX-2) nor androgen receptor (AR). SV40 positive tubulo-acinar tumors seen occasionally in the kidney, did not stain for synaptophysin nor NSE.

The orphan nuclear receptor constitutive active/androstane receptor is essential for liver tumor promotion by phenobarbital in mice.

Hepatocellular carcinoma (HCC) is known to progress through a step often called tumor promotion. Phenobarbital (PB) is the prototype of nongenotoxic cacinogens that promote HCC in rodents. The molecular target of PB to elicit the promotion has been the subject of intense investigations over the last 30 years since it was discovered. The nuclear receptor constitutive active/androstane receptor (CAR) is activated by PB as well as by various other xenobiotics such as therapeutic drugs and environmental pollutants. CAR activation results in the transcriptional induction of numerous hepatic genes including those that encode xenobiotic-metabolizing enzymes such as a set of cytochrome P450s. In addition to PB, many CAR activators are nongenotoxic carcinogens, but the role of CAR in liver tumor promotion remains unexplored. Using Car(-/-) mice, we have here examined tumor promotion by chronic treatment with PB in drinking water after tumor initiation with a single dose of the genotoxic carcinogen diethylnitrosamine. None of the Car(-/-) mice developed either eosinophilic foci or advanced liver tumors, whereas all Car(+/+) mice developed HCC and/or adenoma by 39 weeks. The results indicate that CAR is the molecular target of promotion by PB and that activation of this receptor is an essential requirement for liver tumor development.

Boron supplementation inhibits the growth and local expression of IGF-1 in human prostate adenocarcinoma (LNCaP) tumors in nude mice.

Prostate-specific antigen (PSA) is a serine protease and one of the most abundant proteins secreted by the human prostate epithelium. PSA is used as a well-established marker of prostate cancer. The involvement of PSA in several early events leading to the development of malignant prostate tumors has made it a target for prevention and intervention. It is thought that PSA cleaves insulin-like growth factor binding protein-3 (IGFBP-3), providing increased local levels of IGF-1, leading to tumor growth. Separately, there are data that suggest an enzymatic regulatory role for dietary boron, which is a serine protease inhibitor. In this study we have addressed the use of boric acid as a PSA inhibitor in an animal study. We have previously reported that low concentrations (6 ug/mL) of boric acid can partially inhibit the proteolytic activity of purified PSA towards a synthetic fluorogenic substrate. Also, by Western blot we have followed the degradation of fibronectin by enzymatically active PSA and have found significant inhibition in the presence of boric acid. We proposed that dietary supplementation with boric acid would inhibit PSA and reduce the development and proliferation of prostate carcinomas in an animal model. We tested this hypothesis using nude mice implanted subcutaneously with LNCaP cells in Matrigel. Two groups (10 animals/group) were dosed with boric acid solutions (1.7, 9.0 mgB/kg/day) by gavage. Control group received only water. Tumor sizes were measured weekly for 8 weeks. Serum PSA and IGF-1 levels were determined at terminal sacrifice. The size of tumors was decreased in mice exposed to the low and high dose of boric acid by 38% and 25%, respectively. Serum PSA levels decreased by 88.6% and 86.4%, respectively, as compared to the control group. There were morphological differences between the tumors in control and boron-dosed animals, including a significantly lower incidence of mitotic figures in the boron-supplemented groups. Circulating IGF-1 levels were not different among groups, though expression of IGF-1 in the tumors was markedly reduced by boron treatment, which we have shown by immunohistochemistry. These data indicate that low-level dietary boron supplementation reduced tumor size and content of a tumor trophic factor, IGF-1. This promising model is being evaluated in further studies.

A grading scheme for the assessment of proliferative lesions of the mouse prostate in the TRAMP model.

To improve the precision and consistency of experimental results, we have developed a scoring system for proliferative epithelial lesions in the mouse prostate based on histological growth patterns observed in individual lobes. Severity of proliferative lesions was divided into 6 categories; the grade of the most advanced lesion was identified for each lobe and its distribution estimated semiquantitatively. A numerical score combining grade and distribution of the most advanced lesion in each lobe was assigned and termed the "distribution-adjusted lesion grade"; the mean of these scores was calculated for each treatment group. Using this grading scheme, we assessed lesion development in ad libitum-fed and 20%-diet-restricted groups of TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice that were started on study at 7 weeks of age and sacrificed when 11 and 20 weeks old. The anterior, dorsal, lateral, and ventral prostate lobes showed clear reductions in lesion severity in diet-restricted TRAMPS at 11 and 20 weeks. This method for scoring the epithelial pathology of the prostate in the TRAMP model with minimal to severe proliferative lesions utilizes the natural history of lesion development for assessing the effects of chemical and dietary interventions.

Slowing tumorigenic progression in TRAMP mice and prostatic carcinoma cell lines using natural anti-oxidant from spinach, NAO--a comparative study of three anti-oxidants.

The TRAMP model and human prostatic cancer (PCA) cell lines DU145 and PC3 are useful forchemopreventive studies. We compared the efficacy of 3 anti-oxidants [a water-soluble natural anti-oxidant. NAO (200 mg/kg). found in spinach leaves; epigallocatechin-3 gallate, EGCG (200 mg/kg), a major green tea polyphenol; and N-acetylcysteine, NAC (125 mg/kg)] plus vehicle in slowing spontaneous tumorigenic progression in TRAMP and wild-type male mice. Sacrifices occurred on weeks 5, 9, and 13. Prostatic histopathology and oxidative-stress blood markers were evaluated. Hyperplasias were ranked by a combination of severity grade and distribution (focal, multifocal, and diffuse). The effectivity of each tested compound in reducing the severity/focalness of hyperplasia varied from lobe to lobe. NAO exerted a significant effect on the dorsal and lateral lobes; NAC, on the anterior and ventral lobes, and EGCG, on the ventral lobe. When the most severe hyperplasia in all 4 lobes of TRAMPs was evaluated, only NAO reduced hyperplasia at weeks 9 and 13. Plasma peroxide levels in TRAMPs were reduced following oral administration of NAO or NAC for 13 weeks; EGCG only slightly reduced these levels. In NAO-treated DU 145 and PC3 PCA cells, inhibition of cellular proliferation occurred in a dose-dependent manner, increasing numbers of G1 cells and reducing ROS levels. The anti-oxidative and antiproliferative properties of NAO may explain its efficacy in slowing the spontaneous prostatic carcinogenic process in the TRAMP and its effects in the cell lines.

Evaluation of carcinogenic responses in the Eker rat following short-term exposure to selected nephrotoxins and carcinogens.

This study examined the response of the Eker rat to nephrotoxic compounds and to genotoxic nonrenal carcinogens. Groups of male Eker rats received either no treatment; a vehicle treatment; treatment with a noncarcinogenic nephrotoxin (aluminum nitrilotriacetate, 2 mg/kg/day of aluminum, intraperitoneally, 3 days per week or cyclosporine A, 30 mg/kg/day, orally by gavage, 7 days/week); or treatment with a genotoxic nonrenal carcinogen (furan, 8 mg/kg/day, orally by gavage, 5 days/week or 2,4-diaminotoluene, 6.5 mg/kg/day, orally by gavage, 7 days/week or 2-nitropropane, 89 mg/kg/day, orally by gavage, 3 days/week). Duration of treatment was 4 and/or 6 months. Tissues from the Eker rats were evaluated microscopically and numbers of proliferative renal lesions were counted. Administration of nephrotoxic compounds (Al-NTA and cyclosporine) significantly increased the number of preneoplastic and neoplastic renal lesions in the Eker rat compared to concurrent vehicle controls. The genotoxic nonrenal carcinogens had no consistent effect on numbers of preneoplastic or neoplastic renal lesions and did not produce neoplasms in the expected target organ (liver).

Quantitation of the cancer process in C57BL/6J, B6C3F1 and C3H/HeJ mice.

This study examines growth alterations in liver foci and tumor development as a basis for the different susceptibility in hepatocarcinogenesis found among different strains of mice. Male C57, B6C3F1, and C3H mice treated with a single dose (1 mg/kg) of N,N-diethylnitrosamine (DEN) at 15 days of age and followed up to 12 months displayed a strain-dependent (C3H > B6C3F1 > C57) increase in incidence, number, volume fraction, and size of foci and macroscopic lesions (masses). DEN-treated mice exhibited a time-dependent increase in foci size but not in foci number. Phenobarbital (PB) treatment (500 ppm) in the drinking water starting 2 weeks after DEN-initiation did not affect the incidence or number of masses and foci. In all 3 strains, the bromodeoxyuridine labeling index in foci correlated with foci growth, supporting the major role of cell proliferation in foci growth. Measurements of apoptosis by morphological criteria with H&E staining suggest that intrafocal apoptosis may be a late event preventing foci growth and possibly also promoting focal cell selection, whereas extrafocal apoptosis may facilitate clonal growth by removing adjacent normal cells. The onset of conversion of foci to masses also correlated with strain susceptibility to hepatocarcinogenesis.