Influences of different polychlorinated biphenyls on cytocidal, mitoinhibitory, and nodule-selecting activities of N-2-fluorenylacetamide in rat liver

The influences of different polychlorinated biphenyl (PCB) isomers and congeners on distinct hepatotoxic responses to the hepatocarcinogen N-2-fluorenylacetamide [(2-FAA) CAS: 53-96-3] were examined in F344 rats. Cytocidal toxicity of 2-FAA (25-400 microM), determined by lactate dehydrogenase release during 20 hours in primary monolayer cultures of isolated rat hepatocytes, was reduced by in vivo pretreatment with either phenobarbitone [(PB) CAS: 50-06-6] or 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP), a PB-type PCB inducer. However, cytocidal toxicity of 2-FAA was substantially potentiated by either 3-methylcholanthrene [(MCA) CAS: 56-49-5] or 3,3',4,4'-tetrachlorobiphenyl [(TCBP) CAS: 32598-13-3], an MCA-type PCB. In the same cell culture assays, all four pretreatments similarly reduced cytocidal toxicity of N-hydroxy-N-2-fluorenylacetamide (0.32-32 microM; CAS: 53-95-2). By comparison, pretreatments with either the PB-type or MCA-type PCB's (50-200 mumol/kg) diminished mitoinhibitory toxicity of 2-FAA in vivo, as measured by hepatic regenerative growth and hepatocyte labeling indices 7 days after partial hepatectomy (PH) in rats given 3 consecutive daily doses of 2-FAA (20/mg/kg/day) before PH. This regimen of 2-FAA and PH promoted rapid selective growth of gamma-glutamyltranspeptidase-positive (gamma-GT+) nodules at 2 and 4 weeks after PH in rats previously given an initiating hepatocarcinogen, diethylnitrosamine [(DENA) CAS: 55-18-5]. However, various PCB's, including 2,2',4,4',5,5'-HCBP, 3,3',4,4'-TCBP, 2,2',4,4'-TCBP, 2,2',5,5'-TCBP, and the commercial mixture Aroclor 1254, each given as a single dose of 50 mumol/kg by gavage 10 days after DENA and 7 days before 2-FAA, all reduced the size of 2-FAA-selected gamma-GT+ nodules during the 4-week period after PH. These results indicate that, in spite of predictable inducer-specific opposite influences of different types of PCB's on cytocidal toxicity of 2-FAA, all PCB's similarly reduce nodule selection by 2-FAA in initiated livers. Reduced growth of 2-FAA-selected nodules correlated with the consistent ability of all PCB's to enhance regeneration of liver mass after 2-FAA and PH.     

The activities of 2,2',5,5'-tetrachlorobiphenyl, its 3,4-oxide metabolite, and 2,2',4,4'-tetrachlorobiphenyl in tumor induction and promotion assays

Sensitive assays for the induction of lung adenomas in A/J miceor skin papillomas in SENCAR mice failed to show activity foreither 2,2',5,5'-tetrachlorobiphenyl or 2,2',5,5'-tetrachlorobiphenyl3,4-oxide. Injections of the 3, 4-oxide into preweanling A/Jmice caused considerable mortality, whereas the parent hydrocarbondid not. Both 2,2',5,5'- and 2,2',4,4'-tetrachlorobiphenyl showedpromoting activity for hepatic -glutamyl transpeptidase-positivefoci initiated in rat liver by treatment with diethylnitrosamine.The promoting activity of 2,2',4,4'-tetrachlorobiphenyl was10-fold greater than that of the 2,2',5,5'-isomer.     

Enhancing effect of inducers of liver microsomal enzymes on induction of hyperplastic liver nodules by N-2-fluorenylacetamide in rats.

The effects of inducers of liver microsomal enzymes on the induction of hyperplastic liver nodules by N-2-fluorenylacetamide (2-FAA) were studied in male F344 rats. The rats were fed a diet containing 200 ppm 2-FAA for 2 weeks and then given test chemicals for the following 8 weeks. Partial hepatectomies were performed at the end of the third week of the experiment. The compounds were tested, and their concentrations (in ppm) in the diet were as follows: 1,000 polychlorinated biphenyls (PCB), 500 PCB, 500 PCB plus 70 3-methylcholanthrene (MCA); 500 PCB plus 500 phenobarbital, 70 MCA, 500 phenobarbital, 500 phenobarbital plus 500 beta-naphthoflavone, and 2,500 3-(3,5-dichlorophenyl)-5,5-dimethyloxazoline-dione-2,4 (DDOD). All experimental groups developed significantly greater numbers and total areas of hyperplastic nodules than did the controls. In groups treated similarly with the test chemicals but without partial hepatectomy, the numbers and total areas of hyperplastic nodules were significantly less than those in the experimental groups with partial hepatectomy. Administration of test chemicals except 2-FAA to partially hepatectomized rats did not induce hyperplastic nodules. The present results showed the early detection of the enhancing effect on induction of hyperplastic liver nodules by a system consisting of the following three procedures: 1) 2-FAA feeding; 2) administration of test chemicals, and 3) partial hepatectomy during administration of test chemicals. In this system DDOD enhanced the induction of hyperplastic nodules of the liver.     

Effects on intercellular communication in human keratinocytes and liver-derived cells of polychlorinated biphenyl congeners with differing in vivo promotion activities

Several purified polychlorinated biphenyl (PCB) congeners with differing toxicity/tumor promotional activities in rat liver in vivo were tested for their effects on gap-junctional intercellular communication (GJIC) in cell strains and lines derived from human liver and skin. This in vitro assay is being developed to detect various classes of tumor promoters. The 3-methylcholanthrene (MCA)-type cytochrome P450 inducer and hepatotoxic promoter 3,3',4,4'-tetrachlorobiphenyl was inactive in this assay for all of the cells tested, suggesting this promoter acts by other mechanisms. The phenobarbital-like enzyme inducer and less toxic promoter 2,2',4,4',5,5'-hexachlorobiphenyl inhibited GJIC in both liver and skin cells, whereas the 2,2',5,5'-tetrachlorobiphenyl congener, which does not act as a promoter in rat liver, inhibited GJIC only in the skin cell types and in one of the liver cell strains thought to be of bile duct origin. 2,3,4,4',5-Pentachlorobiphenyl, a mixed (phenobarbital plus MCA) inducer of cytochrome P450, inhibited GJIC in both liver and skin cells, suggesting that it may be a promoter in vivo. The results suggest that GJIC inhibition is a property of PCB congeners with phenobarbital-like enzyme induction capabilities, and that there exist some tissue/cell type differences in sensitivity to these congeners.     

Polychlorinated biphenyls, classified as either phenobarbital- or 3-methylcholanthrene-type inducers of cytochrome P-450, are both hepatic tumor promoters in diethylnitrosamine-initiated rats

The cytochrome P-450 isozymes, cytochrome P-450 MC1 and MC2, purified from rats treated with 3-methylcholanthrene (MC), were found by immunohistochemical staining to be strongly induced in the livers of rats treated with 3,3', 4,4'-tetrachlorobiphenyl (TCBP), while the cytochrome P-450 isozymes, PB1 and PB2, purified from the livers of rats treated with phenobarbital (PB), were shown to be induced in the livers of rats treated with 2,2', 4,4', 5,5'-hexachlorobiphenyl (HCBP). The latter compound also strongly induced NADPH-cytochrome P-450-reductase. Following induction, all 5 enzymes were located preferentially in the centrilobular and midzonal region of the liver acinus. The influence of these polychlorinated biphenyls (PCBs) on diethylnitrosamine (DEN)-initiated hepatocarcinogenesis was investigated by analyzing the evolution of adenosine triphosphatase-deficient focal lesions. Whereas DEN alone produced very few islets, the administration of either PCB congener (150 mumol/kg, i.p., once weekly over a period of 8 weeks) subsequent to DEN treatment (50 ppm in the drinking water, 10 days) strongly enhanced the number of islets as well as the relative volume of liver occupied by islet tissue. These effects were evident, both 1 and 9 weeks, after cessation of PCB treatment. Unexpectedly the less persistent PCB congener, TCBP, showed a much more potent enhancing effect after the 9 weeks recovery period than did (HCBP).     

Effect of dietary retinyl palmitate on the promotion of altered hepatic foci by 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'- hexachiorobiphenyl in rats initiated with diethylnitrosamine

The purpose of this study was to determine the effects of dietaryvitamin A on the tumor promoting effect of 3,3',4,4'-TCB and2,2',4,4',5,5'-HCB in a two-stage rat hepatocarcinogeneis modelwith diethylnitrosamine (DEN, 150 mg/kg) as the initiator. Twoweeks after DEN injection rats were fed a purified diet containingeither 2000 or 100 000 IU of vitamin A in the form of retinylpalmitate. Rats received four biweekly injections of 3,3',4,4'-TCB,2,2',4,4',5,5'-HCB (300 µmol/kg), or both (150 µmol/kgeach) in corn oil (10 ml/kg) for 8 weeks. Control animals receivedvehicle only. Six rats in each group that received no DEN treatmentwere used as additional control animals. Ten days after thelast injection the rats were killed. In rats fed the low retinylpalmitate diet, treatment with 3,3',4,4'-TCB, 2,2',4,4',5,5'-HCBor both compounds lowered hepatic retinyl palmitate content.This effect was prevented by high dietary retinyl palmitatesupplementation in rats treated with 2,2',4,4',5,5'-HCB, butnot 3,3',4,4'-TCB or both com pounds together. Histopathologicalexamination of the liver showed that high dietary retinyl palmitatelessened the severity of hepatocellular necrosis and fatty changesinduced by 3,3',4,4'-TCB alone or in combination with 2,2',4,4',5,5'-HCB.The latter did not cause significant pathological lesions tothe liver. However, high dietary retinyl palmitate was not ableto prevent thymic involution caused by 3,3',4,4'-TCB. The numberand volume of altered hepatic foci were increased by 2,2',4,4',5,5'-HCBand particularly 3,3',4,4'-TCB; no synergistic effect was seen.Supplementation with high dietary retinyl palmitate diminishedthe number and volume of foci. These results show that supplementationwith high dietary retinyl palmit ate protects against hepatocellularnecrosis, fatty changes, and preneoplastic changes induced by3,3',4,4'-TCB as well as against preneoplastic changes inducedby 2,2',4,4',5,5'-HCB. In addition, these two agents did notsynergistically induce preneoplastic changes in DEN-inducedrats.     

Early stages of N-2-fluorenylacetamide-induced hepatocarcinogenesis in male and female rats and effect of gonadectomy on liver neoplastic conversion and neoplastic development

The early stages of N-2-fluorenylacetamide [(2-FAA) CAS: 59-96-3]-induced liver carcinogenesis in inbred F344 male and female rats and the effect of gonadectomy on liver carcinogen biotransformation capability and hepatocarcinogenesis were studied. Feeding of 2-FAA induced more altered hepatocellular foci characterized by exclusion of cellular iron and gamma-glutamyl-transferase activity in male rats than in female rats. At 6-22 weeks after cessation of carcinogen exposure, only males developed liver neoplastic nodules. Liver cytochrome P450, aryl hydrocarbon hydroxylase, and uridine-5'-diphosphateglucuronosyltransferase activity toward p-nitrophenol, but not phenolphthalein, were greater in males than in females. Gonadectomy of males reduced the activities that were greater than in females, whereas none was significantly altered by gonadectomy of females. Gonadectomy of male rats prior to 2-FAA feeding suppressed the induction of both altered foci and neoplastic nodules, whereas in female rats gonadectomy prior to 2-FAA feeding enhanced the induction of foci. Gonadectomy of males after administration of 2-FAA slightly enhanced the persistence of foci at 6 and 12 weeks after removal of carcinogen, but it did not affect their persistence by 22 weeks post carcinogen or the incidence of neoplastic nodules. However, only the males that were gonadectomized after receiving 2-FAA developed hepatocellular carcinomas at 22 weeks. Gonadectomy of females after receiving 2-FAA did not affect the persistence of foci, and no liver neoplasms developed. Thus gonadectomy of male rats, which reduced liver carcinogen metabolism, when done before carcinogen feeding had the greatest effect on hepatocarcinogenesis.     

Breast cancer risk in relation to adipose concentrations of organochlorine pesticides and polychlorinated biphenyls in Long Island, New York.

To assess a possible etiological role of organochlorine compounds in breast cancer development on Long Island, a high-risk region of New York State, concentrations of organochlorine pesticides and polychlorinated biphenyls (PCBs) were measured in the adipose tissue of 232 women with breast cancer and 323 hospital controls admitted to surgery for benign breast disease or non-breast-related conditions. Seven pesticide residues and 14 PCB congeners were assayed via a supercritical fluid extraction method followed by gas chromatography with electron capture detection. After adjustment for age and body mass index, which were strongly correlated with organochlorine levels, adipose concentrations of 1,1-dichloro-2,2-di(4-chlorophenyl)ethylene, total pesticides, and total polychlorinated biphenyls (PCBs) did not differ significantly between cases and controls. The relative abundance of individual pesticide species and PCB congeners was similar in cases and controls. Odds ratios adjusted for age, BMI, hospital, and race gave no evidence of a dose-response for 1,1-dichloro-2,2-di(4-chlorophenyl)ethylene, total pesticides, or total PCBs, whether stratified by estrogen receptor status or not. Breast cancer risk among Long Island residents was not elevated compared with residents of the adjacent New York City borough of Queens. We did not confirm a previously reported association between breast cancer risk and levels of PCB congener 118 (2,3',4,4',5-pentachlorobiphenyl), nor did we observe an association with the most abundant congener 153 (2,2',4,4',5,5'-hexachlorobiphenyl), a strong inducer of phase I enzymes that was reported recently to have estrogenic properties. Only PCB congener 183 (2,2',3,4,4',5',6-heptachlorobiphenyl), which is also an inducer, was significantly associated with risk, with an adjusted odds ratio of 2.0 (95% confidence interval, 1.2-3.4) in women with adipose levels >5.67 ng/g; the biological importance of this observation is unclear without confirmation in additional studies. Although neither the present nor other studies have provided convincing evidence of an association between body burden of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane and PCBs with cancer of the breast, these compounds are rated as "possible" and "probable" human carcinogens, respectively, by the International Agency for Research on Cancer. Investigations of associations with cancer at other sites should be carried out.     

Initiation and selection of resistant hepatocyte nodules in rats given the pyrrolizidine alkaloids lasiocarpine and senecionine

The biological mechanisms by which pyrrolizidine alkaloids contribute to initiation and nodule selection (promotion) steps in hepatic carcinogenesis were studied in male Fischer 344 rats. Lasiocarpine at single or double dosages (up to 80 mumol/kg) delayed hepatic regeneration for at least 8 weeks after partial hepatectomy (PH). This regimen of lasiocarpine and PH had a strong selective influence on the growth of gamma-glutamyltranspeptidase (gamma-GT)-positive hepatocyte nodules in rats previously initiated with diethylnitrosamine. However, both lasiocarpine (up to 80 mumol/kg) and senecionine (up to 160 mumol/kg) were inactive as initiators of gamma-GT-positive nodules in rats exposed to a similar selection regimen consisting of 2-acetylaminofluorene and PH. When lasiocarpine or senecionine was given 12 h after PH, very few nodules were initiated. Lasiocarpine pretreatments reduced the initiating activity of diethylnitrosamine and N-nitrosomethylurea in rats subsequently selected with 2-acetylaminofluorene and PH. Resistant nodules selected with lasiocarpine had the typical resistant nodule phenotype (positive for gamma-GT and epoxide hydrolase) and also lacked pyrrolizidine alkaloid-induced megalocytosis. Lasiocarpine treatment also resulted in small regenerative nodular proliferations of hepatocytes that were distinct from resistant nodules because they were negative for gamma-GT and epoxide hydrolase and unrelated to diethylnitrosamine pretreatments. These studies suggest that the hepatocarcinogenicity of pyrrolizidine alkaloids can be better explained by their strong selection (promotion) influence on initiated hepatocytes, rather than by their very weak initiating activity.     

Neoplastic response of F344 rats and B6C3F1 mice to the polymer and dyestuff intermediates 4,4'-methylenebis(N,N-dimethyl)-benzenamine, 4,4'-oxydianiline, and 4,4'-methylenedianiline

Feeding 4,4'-methylenebis(N,N-dimethyl)benzenamine [CAS: 101-61-1; 4,4'-methylenebis(N,N-dimethylaniline)] to inbred F344 rats increased the incidence of thyroid lesions (hyperplasia, adenomas, and carcinomas) in both sexes, especially in the female rats. 4,4'- Oxydianiline (CAS: 101-80-4) in the diet increased adenomas and carcinomas in the thyroid gland and neoplastic nodules and carcinomas in the liver of male and female F344 rats. In addition to increasing thyroid adenomas in females and hepatocellular adenomas or carcinomas in male and female B6C3F1 mice, 4,4'- oxydianiline increased adenomas of the harderian gland in male and female mice. 4,4'- Methylenedianiline (CAS: 101-77-9) in the drinking water increased neoplasms of the thyroid gland and liver in F344 rats and B6C3F1 mice.     

Dose responses of five hepatocarcinogens for the initiation of rat hepatocarcinogenesis

Using the Solt and Farber model (Nature, 263 (1976) 701), dose-response relationships between initiating agents and the induction of hyperplastic nodules in rat liver were investigated. Male Fischer 344 rats were initiated by a single application of 1 of 5 carcinogens at 3 doses: 200.0, 50.0 and 12.5 mg/kg of diethylnitrosamine (DEN); 1.00, 0.25 and 0.062 mg/kg of aflatoxin B1 (Af-B1); 60.0, 15.0 and 3.75 mg/kg of N-2-fluorenylacetamide (2-FAA); 600.0, 150.0 and 37.5 mg/kg of 3'-methyl-4-dimethylaminobenzene (3'-me-DAB); 40.0, 10.0 and 2.5 mg/kg of dimethylnitrosamine (DMN) and their vehicles. Two weeks after initiation, animals were placed on a 0.02% 2-FAA diet for 2 weeks. Partial hepatectomy was performed at the end of the third week of the experiment. All rats were killed 4 weeks after the initiation, and the hyperplastic nodules of the liver were counted and their areas measured. Dose responses were observed in both numbers and areas of hyperplastic nodules per unit area of sections with all of the 5 carcinogens examined.     

Inhibition of growth by 2,3,7,8-tetrachlorodibenzo-p-dioxin in 5L rat hepatoma cells is associated with the presence of Ah receptor

The role of the Ah receptor in mediating the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in 5L rat hepatoma cells containing TCDD-inducible cytochrome P450IA1 activity and in variants lacking cytochrome P450IA1 and Ah receptor. TCDD inhibited growth of the wild-type 5L cells, but not of the Ah receptor deficient variants. The two strong Ah receptor ligands 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB) and benz[a]anthracene (BA) exerted toxic effects in 5L cells that resembled those of TCDD. The poor Ah receptor ligand 2,2',4,4'-tetrachlorobiphenyl was not toxic in 5L cells. The concentrations of TCDD, 3,3',4,4'-TCB or BA required for the toxic response were similar to those that elicited P450IA1 induction. The present results suggest strongly that interaction with the Ah receptor is a necessary link in the chain of events leading to toxic effects in 5L cells upon exposure to TCDD.     

Survey of various chemicals for initiating and promoting activities in a short-term in vivo system based on generation of hyperplastic liver nodules in rats

The effects of pre- (initiation stage) and post- (promotion stage) administration of 17 chemical carcinogens (including both hepatic and non-hepatic carcinogens), 3 promoters and 2 non-carcinogenic analogs, on the induction of liver hyperplastic nodules were investigated in rats. Test chemicals were administered during the initiation stage after which rats were fed dietary N-2-fluorenylacetamide (2-FAA) for 2 weeks in conjugation with necrogenic CCl4 to enhance production of nodules initiated by test chemicals. Alternatively, effects of test chemicals administered during the promotion phase were evaluated in rats given a combination of dietary 2-FAA for 2 weeks and necrogenic CCl4. This initiation regimen resulted in very few nodules unless a promoter was subsequently used. All chemical carcinogens administered during the initiation stage induced more frequent, larger hyperplastic nodules than did control treatments. However, neither the promoters nor the non-carcinogenic analogs induced hyperplastic nodules if they were administered during the initiation stage. In contrast, hepatocarcinogens and a promoter of hepatocarcinogenesis (phenobarbital) administered in the promotion stage had enhancing (promoting) activity on hyperplastic liver nodules, whereas non-hepatocarcinogens, a promoter for skin carcinogenesis (12-O-tetradecanoylphorbol-13-acetate) and a promoter for urinary bladder carcinogenesis (saccharin) did not. Non-carcinogenic analogs were not active when administered during either the initiation or promotion stages. These findings demonstrated the utility of employing short-term in vivo assays for both initiating and enhancing (promoting) activities of chemicals. By our system, chemicals were classified into 4 main groups, namely, (i) hepatocarcinogens which have both initiating and enhancing activity, (ii) non-hepatocarcinogens with initiating activity only, (iii) promoters of hepatocarcinogenesis which have promoting activity and (iv) non-carcinogens and non liver promoters which did not initiate nor promote nodule development. This is a more discriminating means of assaying carcinogenic activity than is possible with short-term in vitro screening assays for mutagenicity.     

Expression of fibronectin and laminin in the rat liver after partial hepatectomy, during carcinogenesis, and in transplantable hepatocellular carcinomas

The distribution in the F344 rat liver of two extracellular matrix and basement membrane components, fibronectin and laminin, was studied by immunofluorescence. Fibronectin was found diffusely in normal liver lining the sinusoids and in connective tissue surrounding blood vessels and bile ducts; laminin was present predominantly in the basement membranes of blood vessels and bile ducts and was only inconsistently seen lining the sinusoids. After partial hepatectomy (PH), there was a transient decrease of fibronectin in the central and midzone sinusoidal hepatic areas. This decrease was most marked on day 3 after the PH. Carcinogens caused marked changes in the distribution of fibronectin. Large extracellular deposits of fibronectin were seen in areas of oval cell proliferation in livers of rats treated with N-2-fluorenylacetamide (2-FAA) while being fed a choline-deficient diet. In contrast, the nodules that developed in these livers were almost completely devoid of fibronectin staining. Neoplastic nodules produced in rats by cyclic feedings of 2-FAA r by injections of diethylnitrosamine also contained little or no fibronectin. Laminin staining did not change markedly during these treatments, but increased staining was seen associated with the newly formed ductlike structures and oval cells in liver of rats treated with carcinogens. Transplantable hepatomas varied in their fibronectin staining from fibronectin-negative hepatomas to ones with fibronectin staining within or around every tumor cell. Laminin was only found around the vascular structures within the tumors. The presence or absence of fibronectin in hepatomas did not show an obvious correlation to growth rate or metastatic potential of the tumors studied.     

Polybrominated biphenyls as promoters in experimental hepatocarcinogenesis in rats

Female Sprague Dawley rats were fed polybrominated biphenyls(PBB) for six months after a 70% partial hepatectomy and diethylnitrosamineadministration (10 mg/kg body weight) to determine if PBB couldserve as a tumor promoter in a two stage hepatocarcinogenesistest system. Firemaster BP-6, a commercial mixture of PBB, andits major congener, 2,2',4,4',5,5' -hexabromobiphenyl (HBB)were used in this study. Tumor promoting ability was assessedby measuring enzyme altered foci exhibiting gamma glutamyl transpeptidaseactivity. Dietary concentrations of 10 and 100 p.p.m. of themixture of PBB and of HBB were found to be promoters of hepatocarcinogenesis.The mixture of PBB had a greater tumor promoting ability thanHBB.     

Inhalation carcinogenesis of various alkylating agents

A series of earlier studies showed that inhalation exposures of rats to three water-reactive electrophilic compounds produced brisk yields of nasal cancer even when the animals were exposed for only 30 days (6 hr/day X 5 day/wk). In addition, carcinogenic potencies of the compounds appeared to relate to their chemical reactivities as measured by hydrolysis rates. For a further study of this phenomenon, inhalation exposures were conducted with five additional water-reactive compounds: beta-propiolactone [(BPL) CAS: 57-57-8], methylmethane sulfonate [(MMS) CAS: 66-27-3], ethylchloroformate [(ECF) CAS: 541-41-3], dichloroacetyl chloride [(DCAC) CAS: 79-36-7], and propylene oxide [(PO) CAS: 75-56-9] on male Sprague-Dawley rats. The hydrolysis rates of these compounds span 6 orders of magnitude. The compounds were administered for 30 days (6 hr/day X 5 days/wk) with the use of exposure concentrations that were inversely proportional to the respective hydrolysis rates. With this protocol, all compounds except PO (the slowest reacting compound) produced nasal cancer in rats. The concentrations of MMS and BPL employed in the studies produced similar nasal cancer yields, indicating that the carcinogenic potencies of these compounds in rat nasal mucosa were proportional to their hydrolysis rates. The nasal cancer yields of DCAC and ECF were less than expected. DCAC hydrolyzes so rapidly at in vivo temperatures (half-life much less than 0.01 min) that it may not reach target DNA in reactive form. Why the exposures to ECF produced yields of nasal cancer not predicted by its reactivity is currently under investigation. These results combined with our earlier results demonstrate that the carcinogenic potencies of some inhaled reactive electrophilic compounds are related to their hydrolysis rates.     

Hepatic foci of cellular and enzymatic alteration and nodules in rats treated with clofibrate or diethylnitrosamine followed by phenobarbital: their rate of onset and their reversibility

The histologic appearance and cytochemical characteristics of foci of hepatic cellular alteration, hepatic nodules, and hepatocellular carcinomas occurring in male Sprague-Dawley rats treated with the hypolipidemic agent clofibrate (CAS: 637-07-0), with phenobarbital (CAS: 50-06-6), or with diethylnitrosamine [(DENA) CAS: 55-18-5] followed by phenobarbital were studied after treatment periods from 1 month to 2 years. Rats treated with clofibrate revealed foci of cellular alteration that were more often basophilic and occurred slightly sooner (wk 42) than those in untreated controls (wk 60). Of 36 rats that had received 68 or more weeks of continuous clofibrate, 19 had hepatic nodules. Of the 11 nodules examined cytochemically, none was gamma-glutamyltransferase (gamma-GT) positive and 2 were positive to glucose-6-phosphate dehydrogenase (G-6-PD) under oxygen. In rats withdrawn from clofibrate for 16-18 weeks after 68-95 weeks of clofibrate, 0 of 14 had nodules. In several of these rats zones of hepatic scarring were observed, suggesting the reversibility of the nodules. Phenobarbital alone had little effect on the incidence of foci of cellular alteration, although the number of gamma-GT-positive foci was increased. DENA followed by phenobarbital led to the early appearance of foci of cellular alteration (from wk 4), of nodules (from wk 13), and of hepatocellular carcinomas (from wk 26). gamma-GT activity was raised in most of these nodules and carcinomas, while G-6-PD activity was raised in only 3 of 9 nodules but in all 9 carcinomas examined. DENA-phenobarbital given for 13 or 26 weeks followed by withdrawal of phenobarbital for 28 and 26 weeks, respectively, produced an essentially similar pattern of lesions. In view of the growing recognition of the nonspecificity gamma-GT as a marker of carcinogen-initiated foci, the value of G-6-PD (under oxygen) as a marker merits further investigation.     

Splenic fibrosis and sarcomas in F344 rats fed diets containing aniline hydrochloride, p-chloroaniline, azobenzene, o-toluidine hydrochloride, 4,4'-sulfonyldianiline, or D & C red No. 9

In six carcinogenicity bioassays, male and female F344 rats were fed diets containing aniline hydrochloride (CAS: 142-04-1; hydrochloride benzenamide), p-chloroaniline (CAS: 106-47-8), azobenzene (CAS: 103-33-3), o-toluidine hydrochloride (CAS: 636-21-5), dapsone (CAS: 80-08-0; 4,4'-sulfonyldianiline), or D & C red No. 9 [CAS: D85500000; 5-chloro-2-[2-hydroxy-1-naphthalenyl)azo)-4-methylbenzenesulfon ic acid, barium salt]. The rats, from 6 weeks to 2 years old, were given the compounds at two dose levels, the estimated maximum tolerated dose and one-half that dose. In all six bioassays, dose-dependent incidences of splenic sarcomas and fibrosis were seen, with the highest incidences in male rats. Fibrosis occurred in the splenic parenchyma and/or the capsule. Fatty infiltration also was seen in the spleen. Sarcomas appeared to arise in the splenic red pulp or splenic capsule, usually in association with areas of parenchymal and capsular fibrosis and pigmentation. Larger tumors metastasized to the peritoneal cavity and abdominal organs. In some rats there was marked osseous metaplasia when the primary tumor metastasized to peritoneal surfaces. Other, less common, splenic neoplasms included hemangiosarcoma and hemangiopericytoma. Some rats had such extensive peritoneal involvement that the site of origin of their sarcoma was difficult to determine.