Sequential histopathologic analysis of alpha-hexachlorocyclohexane-induced hepatic megalocytosis and adenoma formation in the HPB mouse.

A temporal, gross, and histologic analysis of the livers of male HPB black mice maintained on a diet containing 500 ppm alpha-hexachlorocyclohexane (alpha-HCH) was performed at 1, 3, 4, 8, 14, 21, 30, 33, 44, and 50 weeks. Grossly, progressive liver enlargement was first noticed at 3 weeks, hepatic nodules at 21 weeks, and emaciation at 30 weeks. Histopathologic liver alterations included universal hypertrophy of centrolobular hepatocytes first seen at 1 week and the merging of adjacent megalocytic zones at 3 weeks. At 21 weeks, microadenomata and macroadenomata were seen in 2 of 7 mice. At 30 weeks, adenomata occurred in 7 of 8 mice and at 33, 44, and 50 weeks in 6 of 6, 5 of 5, and 5 of 5 mice, respectively. Individual adenomata were composed of large well-packed cells with basophilic and acidophilic pale-staining or lipid-laden cytoplasm forming disorganized cords of variable thickness. Depending on the stage of development, adenomata were classified into 4 subtypes. Subtype I, the earliest form seen, arose within megalocytic areas and was composed of a small number of megalocytic cells exhibiting loss of polarity. Subtype II was smaller than a liver lobule. Subtype III was larger and at times resulted from the merging of adjacent subtype II nodules. Subtype IV included the largest adenomata, most of which resulted from coalescing smaller sized subtypes. Under the conditions of this experiment, neither hepatocellular carcinoma nor metastases in the lungs were detected. It was concluded that if alpha-HCH-induced hepatocellular adenoma is ever to give rise to hepatocellular carcinoma, this transformation must progress very slowly.

Phenolic antioxidants: Health Protection Branch studies on butylated hydroxyanisole.

Synthetic phenolic antioxidants have been added to foods for decades to retard the autooxidation of lipid that leads to rancidity. The major antioxidants, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), are used in foods world wide. Recent studies suggest that BHA, and perhaps BHT, are carcinogenic to rodents. International efforts, including those at the HPB in Ottawa Canada, have helped place the results of the chronic rodent studies into perspective. It seems likely that the neoplastic effects observed at very high dietary levels of BHA and BHT occur only after effective biological defense mechanisms are overloaded. The renewed interest in the toxicity of phenols is beneficial to an understanding of the complex biological effects of naturally occurring phenolics, including reduction of the levels of reactive oxygen species that are associated with various disease states in an aging human population.

The inhibition of urease and proteases by sodium saccharin.

Sodium saccharin, at concentrations similar to those in the urine of rats fed 1-5% sodium saccharin in their diet, markedly inhibited urease, and 3 proteases in vitro and sodium ion did not appear to play a role in enzyme inhibition. These observations suggest that enzyme inhibition of any of a large number of enzymes may play a role in the tumorigenesis of the urinary bladder by saccharin.

Dietary restriction, cell proliferation and carcinogenesis: a preliminary study.

Four groups of female Swiss Webster mice were given either laboratory chow or a purified (semi-synthetic) diet (AIN-76A) either ad libitum or at 75% of the ad libitum rate for about 30 days. Three tissues, the crypt cells of the jejunum, the dermis and the basal epithelial cells of the esophagus were investigated using [3H]thymidine labelling and by counting mitoses; four other tissues, the alveolar cells of the mammary gland, the crypt cells of the duodenum and colo-rectum, and the transitional cells of the urinary bladder were examined using [3H]thymidine labelling only. In each case dietary restriction led to a reduction of cellular proliferation assessed by these indices. The potential of the approach for the study of the effects of dietary modification on the induction of cancer is discussed.

An 85-day study of butylated hydroxyanisole in the cynomolgus monkey.

Groups of 8 cynomolgus monkeys (Macaca fascicularis) were given 500, 125 and 0 mg/kg body wt butylated hydroxyanisole (BHA) by gavage in corn oil 5 times/week for 20 days, after which the high dose was halved. No significant adverse clinical signs nor abnormal fibroscopic observations were noted before the experiment was terminated at 85 days. Blood levels of glucose, albumin, chloride, red blood cells (RBCs), hemoglobin (HGB), hematocrit (HCT) and mean corpuscular hemoglobin (MCH) were altered in a dose related manner but the altered values were well within normal ranges reported for this species. While histopathology showed no treatment related effects, the mitotic index was elevated 1.9-fold in the distal esophagus of monkeys in the high but not in the low dose group. Liver weights were increased in a dose related manner but liver monooxygenases, with the exception of decreased ethoxyresorufin deethylase activity, were all within normal limits. BHA given orally to monkeys at about the maximum tolerated dose failed to induce the massive changes noted with rats given 2% dietary BHA.

Effects of antioxidants on aflatoxin-induced hepatic tumors in rats.

Female Wistar rats pretreated with Aflatoxin B1 (AFB) were administered reduced glutathione, butylated hydroxytoluene, methionine or ascorbic acid on a daily basis, p.o., for 8 months. None of the treatments produced a decrease in incidence or size of hepatic nodules. While there was some evidence that ascorbic acid reduced the incidence of cystic cholangioma, the ascorbic acid and methionine treatment groups also contained significantly fewer animals surviving to the 26-month sacrifice. The lack of effect of glutathione is not consistent with previous work showing a marked glutathione dependent regression of AFB-induced hepatocellular carcinoma.

In vitro cytotoxicity of polychlorinated biphenyls (Aroclors 1016, 1242, 1254 and 1260) and their effect on phospholipid and neutral lipid composition of Chinese hamster ovary (CHO-K1) cells.

Cytotoxicity of 4 Aroclors (1016, 1242, 1254 and 1260) was compared in Chinese hamster ovary (CHO-K1) cells in Ham's F-12 medium. When parameters of toxicity were cell numbers or tissue protein, 50% lethality occurred at Aroclor concentrations between 30 and 45 ppm. An in vitro clonal assay with CHO-K1 cells was a sensitive indicator of cytotoxicity of the polychlorinated biphenyls (PCBs). From EC50 values (concentration that allowed 50% survival of formed colonies), cytotoxicity was lower with Aroclor 1016 (32 ppm) and higher with Aroclors 1254 (27 ppm) and 1260 (28 ppm). In cells exposed 24 h to a marginally cytotoxic dose (20 ppm) of each Aroclor, phospholipid (PL) thin-layer chromatography (TLC) showed an increase in phosphatidylcholine (PC) and a decrease in phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG). Neutral lipid (NL) TLC of cells given Aroclors 1242, 1254 or 1260 showed a 3-4-fold increase in triglyceride (TG) and a similar reduction in cholesteryl esters (CE); in contrast to Aroclor 1016 which produced no change in TG and a smaller (2-fold) reduction in CE. Cholesterol and free fatty acid fractions were unaffected by any of the Aroclors. The TG:PL ratio remained unchanged in cells given Aroclor 1016, but increased 3-4-fold with Aroclors 1242, 1254, or 1260. Compared to total values in the untreated controls, CHO-K1 cells contained less neutral lipid and more phospholipid only with Aroclor 1016. These results support the concept that differences in the behavior of Aroclor 1016 are related to its PCB composition. Changes in membrane PL and NL components, observed at marginally cytotoxic levels of each Aroclor, provided further evidence that the PCBs may affect membrane integrity and associated metabolic functions.

A 13-week feeding study of butylated hydroxyanisole: the subsequent regression of the induced lesions in male Fischer 344 rat forestomach epithelium.

Feeding butylated hydroxyanisole (BHA) to male Fischer 344 rats at concentrations of 2, 0.5, 0.25, 0.1 and 0% for 13 weeks led to proliferative lesions developing in the forestomach epithelium of the 2%-treated rats but not in other groups. The [methyl-3H]thymidine labelling index was raised in the 2%- and 0.5%-treated groups and showed an apparent no observable effect level at 0.25%. Within 1 week after withdrawal of BHA the labelling indexes in all treated groups returned to near the values in the controls. The induced mucosal lesions, however, reverted more slowly and even after 9 weeks on the basal diet, the stratified squamous epithelium along the lesser curvature, was still slightly thicker than the control. There were multilayered basal cell processes in the lamina propria with connections to the basal cell layer. The possible significance of these results to the ultimate development of cancer is discussed.

Long-term effects of Aroclor 1254 (PCBs) on plasma lipid and carnitine concentrations in rhesus monkey.

Sixty-seven female rhesus monkeys (Macaca mulatta) were orally dosed daily for 152 weeks with 0, 5, 20, 40, and 80 micrograms Aroclor 1254 (PCB)/kg body wt. Blood polychlorinated biphenyl (PCB) concentrations were highly positively correlated (r = 0.92, P < 0.001) with doses of PCB administered. A comprehensive analysis of plasma lipids/lipoproteins revealed a PCB-associated increase in plasma triglycerides and decreases in plasma total cholesterol, high-density lipoprotein cholesterol (HDL-chol), very-low plus low-density lipoprotein cholesterol (VLDL+LDL-chol), and total carnitine (which is involved in fatty acid metabolism). All of the lipid/lipoprotein changes were significantly (P < or = 0.05) correlated with blood PCB concentration. These data, obtained after 152 weeks of continuous daily exposure of a primate model to PCB support a causal relationship between plasma lipid changes and PCB intake. Previously, causality has been refuted on the premise that the commonly observed elevation of triglycerides with increasing concentration of blood PCB is a reflection, not of PCB dose, but of the partitioning of PCB between tissues (adipose) and blood in proportion to the blood lipid present. The mechanism of the plasma lipid changes was not investigated in this study but the altered lipid/lipoprotein pattern is discussed with respect to known cardiovascular risk profiles.

A carcinogenesis reversibility study of the effects of butylated hydroxyanisole on the forestomach and urinary bladder in male Fischer 344 rats.

A reversibility study was initiated to determine if the length of feeding with 2% butylated hydroxyanisole (BHA) altered the incidence of forestomach lesions observed after a 24-month observation period. Groups of male Fischer 344 rats were fed 2% BHA for 0, 3, 6, 12, and 24 months and then the basal diet for the completion of the 24-month experimental period. Subgroups were serially sacrificed for histopathological examination and [methyl-3H]thymidine radioautography at the time when each group of animals was transferred to the basal diet and also at 15 months. The results showed that except for carcinomas and some epithelial downgrowths, cellular proliferation, measured by radioautography in the epithelium lining the greater and the lesser curvature of the forestomach, remained dependent on the continuous presence of 2% BHA for, at least, 12 months. Superficial hyperplasias, inflammatory lesions and many of the papillomas regressed after cessation of treatment at 12 months. The epithelial downgrowths did not appear to enlarge after the BHA was withdrawn. The squamous cell carcinomas occurred in almost identical yields whether the rats were fed 2% BHA for 12 months and then returned to the basal diet for 12 months or received 2% BHA continuously for 24 months. It is shown here that at several times, 2% BHA stimulated the [methyl-3H]thymidine labelling index of the transitional epithelium of the urinary bladder and that at 3 months the no observed effect level was greater than 0.5% BHA. The significance of the studies on the forestomach and bladder epithelia are discussed. It is concluded that the lesions induced by BHA are most unlikely to be relevant to humans exposed to much lower levels of BHA.

Short-term effects of various phenols and acids on the Fischer 344 male rat forestomach epithelium.

A series of phenols and acids was fed to rats for 9 days to determine effects on the [methyl-3H]thymidine labelling index and the histological appearance of the forestomach. A variety of proliferative effects in the rat forestomach were observed which paralleled changes in the [methyl-3H] thymidine labelling index. The 3-tert-butyl isomer of butylated hydroxyanisole (BHA) was as effective as the food grade mixture. In the 4-hydroxybenzoic acid ester series, activity was not found either with the free acid or the methyl ester. With the ethyl, n-propyl and n-butyl esters activity in the perfundic region of the forestomach increased with alkyl chain length, the n-butyl ester being nearly as effective as BHA. In contrast, 4-methoxyyphenol and propionic acid demonstrated their greatest effects in the midregion of the forestomach, the action of propionic acid not being apparent until 21 or 27 days of treatment. It was also found that after a 9-day treatment involving coadministration of BHA and acetylsalicyclic acid, the overall effect of the antioxidant on the forestomach was greatly reduced.

Short-term pathological and proliferative effects of butylated hydroxyanisole and other phenolic antioxidants in the forestomach of Fischer 344 rats.

Food grade butylated hydroxyanisole (BHA) when incorporated in the diet and fed to male Fischer 344 rats for 9 or 27 days induced proliferative squamous epithelial changes in the lesser curvature of the forestomach proximate to the glandular stomach. These changes were assessed histopathologically and by [methyl-3H]thymidine radioautography. It was shown that BHA mixed dry into powdered diet, incorporated into the diet in corn oil, or in a pelleted diet, induced similar effects. When levels of 2%, 1%, 0.5%, 0.25%, 0.1% and 0% BHA were incorporated in rat diet for 9 days, the proliferative effect appeared to show a no effect level at 0.25% based on the [methyl-3H]thymidine-labelling index. Other food use antioxidants, namely butylated hydroxytoluene or tertiary butylhydroquinone, induced a lesser response than BHA at the maximum dose employed in the study. Propyl gallate was without effect. Propyl-4-hydroxybenzoate, a food use phenol, on the other hand, induced a less pronounced response than BHA but was more effective than the other antioxidants. Because increased cellular proliferation often provides an optimal milieu for tumor formation, it is suggested that these observations may be relevant to rat forestomach tumors induced by BHA.

Effects of rifampin pretreatment on hepatic parameters in the rabbit.

Rifampin pretreatment in the rabbit caused a selective induction of hepatic parameters resembling in some respects phenobarbital induction. Concomitant with induction, a transient selective inhibition of hepatic parameters was also observed. This two-fold effect of rifampin offers an explanation for the discrepancy surrounding the dosage and species differences in hepatic induction reported in the literature.

Monooxygenase-mediated metabolism and binding of ethylene thiourea to mouse liver microsomal protein.

Ethylenethiourea (ETU), a metabolite and degradation product of the ethylenebisdithiocarbamate fungicides, is a substrate for the flavin-dependent monooxygenase (FMO), a microsomal NADPH requiring enzyme that can oxidize ETU, as well as for the cytochrome P-450 enzyme system. The present study shows that the mouse metabolises ETU preferentially via the FMO system. FMO activity decreases as male, but not female mice, increase in age to 30 weeks. This difference in activity is reflected in decreased overall metabolism of ETU and in a decreased FMO-mediated binding of radiolabelled ETU to mouse liver microsomal protein. The rapid metabolism of ETU by the FMO system may contribute to the lack of acute toxicity and teratogenicity exhibited by the mouse relative to the rat. However, the FMO-mediated binding of ETU metabolites to mouse liver protein is consistent with the chronic hepatotoxicity exhibited by ETU in this species.

In vivo and in vitro binding of alpha- and gamma-hexachlorocyclohexane to mouse liver macromolecules.

alpha-Hexachlorocyclohexane (alpha-HCH) but not gamma-HCH, produces a strong neoplastic response in HPB strain mouse liver. In vivo and in vitro binding studies with 14C-labelled HCH isomers showed no preferential binding of the alpha-HCH isomer to protein or DNA. Moreover, the binding of both isomers to DNA in vivo or in vitro occurred only at very low levels, a result consistent with the lack of mutagenic activity associated with these compounds. The results suggest that the neoplastic response observed with alpha-HCH results from a non-genotoxic mechanism.

Effects of deoxynivalenol (vomitoxin) on the humoral and cellular immunity of mice.

Sublethal doses (0.00, 0.25, 0.50 and 1.00 mg/kg b.w./day) of vomitoxin (deoxynivalenol; DON) were studied for their effects on humoral and cellular immunity and serum proteins of inbred, male Swiss Webster mice in a series of 4 separate experiments. Vomitoxin was added to basal diet (less than the detection limit, i.e., less than 0.05 micrograms of vomitoxin per g of feed) and administered to mice for 5 weeks beginning at 21 days of age. Mice in experiment 2 were fed the basal diet for 40 days in addition to the 5-week treatment with vomitoxin. The 1.00 mg/kg dose of vomitoxin resulted in a statistically significant reduction in the serum levels of alpha 1 and alpha 2-globulins, an increase in total serum albumin, and a reduction in feed consumption and body weight gain compared to the control group. The 0.50 mg/kg dose of vomitoxin resulted in significantly reduced serum levels of alpha 2- and beta-globulins while a significant reduction of feed consumption was evident only during Week 4. Similarly, body weight gain in this group of mice was significantly reduced during Week 2 but increased to normal levels during Week 3 and remained parallel to the control for Week 4 and 5. Both levels (0.50 and 1.00 mg/kg) of vomitoxin resulted in a reduced, dose-related, time-to-death interval following a challenge with L. monocytogenes and increased proliferative capacity of splenic lymphocyte cultures stimulated with the phytohemagglutinin P (PHA-P) mitogen compared to the control group of mice. The 0.25 mg/kg dose of vomitoxin did not have any significant effects on the parameters studied. A reasonable estimation of a 'no effect' level for immunologic effects in mice based on these and previous immunological studies would seem to be between 0.25 and 0.50 mg/kg b.w./day.

International Commission for Protection Against Environmental Mutagens and Carcinogens. Oxidative DNA damage--the effects of certain genotoxic and operationally non-genotoxic carcinogens.

A wide variety of oxidative DNA lesions are commonly present in untreated human and animal DNA. One of these lesions, 8-hydroxydeoxyguanosine, has been shown to lead to base mispairing (mutation) on DNA replication. Other lesions remain to be investigated in this respect. Oxidative DNA lesions on cell replication may, in appropriate circumstances, lead to proto-oncogene activation. Oxidative DNA damage, on fixation, may also lead to cytotoxicity followed by regenerative proliferation. The probable or possible importance of oxidative DNA damage is reviewed for various classes of carcinogens and natural processes, including metal ions, high-energy radiation, miscellaneous chemicals, tumor-promoting agents, polyhydroxyphenols/quinones, lipid metabolism, peroxisome proliferators and thyroid function. It is concluded that although the evidence needs considerable strengthening in many of these examples, the available information indicates the potential importance of oxidative DNA damage in the induction of tumors by these agents. It is also possible that non-cancerous degenerative diseases associated with aging are the result of the accumulation of lesions resulting from unrepaired oxidative DNA damage.

Early indicators of potential neoplasia produced in the rat forestomach by non-genotoxic agents: the importance of induced cellular proliferation.

Forestomach neoplasia induced by the apparently non-genotoxic carcinogens, butylated hydroxyanisole and propionic acid, appears to arise by way of sustained high levels of cellular proliferation. Several other inducers of enhanced cellular proliferation, or the consequential incidence of hyperplastic lesions, have been identified in the rodent forestomach but the requisite carcinogenicity bioassays remain undone. In other tissues, such as the male rat kidney, the rodent thyroid follicular cell and the bladder epithelium, there is also evidence supporting the concept that sustained enhanced cellular proliferation may be an important early marker for non-genotoxic carcinogens. This reaction is, however, not likely to be the only marker necessary for the identification of non-genotoxic carcinogens.

Induction of rat hepatic monooxygenase activity by polychlorinated diphenyl ethers.

Polychlorinated diphenyl ethers are recognized environmental contaminants. Twelve of these compounds were tested for their ability to induce liver cytochrome P-450 and monooxygenase activities in Sprague-Dawley rats. All the compounds increased P-450 levels or increased monooxygenase activities in a manner resembling 3-methylcholanthrene, phenobarbital or a combination of both (mixed). The responses obtained resembled those of the polychlorinated biphenyls, some of which are known to be toxic.

Subchronic toxicity study of domoic acid in the rat.

Male and female Sprague-Dawley rats were dosed by gavage for 64 days with 0, 0.1 or 5 mg/kg/day domoic acid. Treated animals showed no clinical abnormalities. Terminal values in haematology and clinical chemistry did not reveal differences between treated and control groups. Findings in histopathology and immunohistochemistry were unremarkable. The 24-hr urinary excretion rate for domoic acid determined at three time points was approximately 1.8% of the dose and remained unchanged during the study.