Ppm Phenobarbital Research Articles

Aldehyde dehydrogenase as a marker for chemically initiated mouse liver tumors

The induction of an aldehyde dehydrogenase isozyme (B-ALDH), which uses aromatic aldehydes such as benzaldehyde as substrate and NADP as a cofactor was examined in normal liver and tumors of B6C3F1 male mice. The tumors were induced by either 15 or 45 ppm diethylnitrosamine (DENA) in the drinking water for 4 weeks. In some cases, the DENA treatment was followed with 500 ppm phenobarbital (PB) in the drinking water. DENA increased the percent of the mice that showed B-ALDH activity in their livers 14 weeks after termination of DENA treatment. The B-ALDH activity was confined mainly in the centrilobular region. Fifty weeks after the termination of the DENA treatment the percent of animals with B-ALDH in their livers was not different from control mice. However, a larger number of the tumors exhibited B-ALDH activity. These studies show that B-ALDH is a good histochemical marker for mouse liver tumors and may be useful as a marker for the effect of chemical carcinogens in this species.

The effects of selenium on the emergence of aflatoxin B 1-induced enzyme-altered foci in rat liver

The effects of selenium on the emergence of aflatoxin B 1 (AFB 1)-induced enzyme-altered foci were studied in male Sprague-Dawley rats. Animals were fed a selenium-deficient diet and supplemented with 5.0, 2.0, and 0.2, or 0 ppm selenium in drinking water for 3 weeks prior to initiation with 2.0 μmol/kg AFB 1. After a 1-week period of selenium normalization, the animals were placed on a diet of ordinary rat chow, and were administered a promoting regimen of 500 ppm phenobarbital in drinking water for 1 week, after which time each rat received a two-thirds partial hepatectomy. The promoting regimen of phenobarbital in tap water was then reduced to 100 ppm and continued for 7 weeks. Subsequently, the rats were sacrificed, their livers excised, and fresh frozen sections prepared and stained histochemically to demonstrate areas of γ-glutamyl transpeptidase (GGT) activity. Selenium supplementation was observed to diminish the induction of GGT-positive foci, especially at the 5.0-ppm level. These data suggest that selenium is able to protect against the hepatocarcinogenic effects of AFB 1 in the rat, and that the enzyme-altered foci bioassay may be a useful technique in assessing the interaction of selenium on the process of hepatocarcinogenesis.

The Effects of Selenium on the Emergence of Aflatoxin B1-Induced Enzyme-Altered Foci in Rat Liver

The Effects of Selenium on the Emergence of Aflatoxin B1-Induced Enzyme-Altered Foci in Rat Liver. MILKS, M. M., WILT, S. R., ALI, I. I., and COURI, D. (1985). Fundam. Appl. Toxicol. 5, 320–326. The effects of selenium on the emergence of aflatoxin B1 (AFB1)-induced enzyme-altered foci were studied in male Sprague-Dawley rats. Animals were fed a selenium-deficient diet and supplemented with 5.0, 2.0, and 0.2, or 0 ppm selenium in drinking water for 3 weeks prior to initiation with 2.0 μmol/kg AFB1. After a 1-week period of selenium normalization, the animals were placed on a diet of ordinary rat chow, and were administered a promoting regimen of 500 ppm phenobarbital in drinking water for 1 week, after which time each rat received a two-thirds partial hepatectomy. The promoting regimen of phenobarbital in tap water was then reduced to 100 ppm and continued for 7 weeks. Subsequently, the rats were sacrificed, their livers excised, and fresh frozen sections prepared and stained histochemically to demonstrate areas of γ-glutamyl transpeptidase (GGT) activity. Selenium supplementation was observed to diminish the induction of GGT-positive foci, especially at the 5.0-ppm level. These data suggest that selenium is able to protect against the hepatocarcinogenic effects of AFB1 in the rat, and that the enzyme-altered foci bioassay may be a useful technique in assessing the interaction of selenium on the process of hepatocarcinogenesis.

Trichloroethylene effects on the formation of enzyme-altered foci in rat liver.

The initiating and promoting effects of trichloroethylene in rat liver were investigated using the enzyme-altered foci bioassay. The incidence of gamma-glutamyl transpeptidase (GGT)-positive foci was used as an early histochemical marker of putative preneoplastic hepatocytes. A single PO dose of trichloroethylene (490 mg/kg) was administered in corn oil to rats which had been partially hepatectomized 24 h previously. Three days following gavage with the chlorinated hydrocarbon the rats were promoted with an 8-week regimen of 500 ppm phenobarbital in drinking water. This protocol is known to induce enzyme-altered foci in the livers of animals which have received an initiating dose of a genotoxic carcinogen. Trichloroethylene was not found to induce GGT-positive foci under these conditions. Additionally, groups of rats were partially hepatectomized, initiated with N-nitrosodiethylamine (30 mg/kg; PO) and administered five times weekly doses of 200 mg trichloroethylene per rat in order to investigate the promoting activity of the chlorinated hydrocarbon in rat liver. No significant promoter effects were observed with trichloroethylene, although the results in this case were somewhat equivocal. The findings of these investigations are taken as partially supportive of an epigenetic, cytotoxic mechanism of tumorigenic action of trichloroethylene.

Perturbation of calcium homeostasis by CCl4 in rats pretreated with chlordecone and phenobarbital.

Male Sprague-Dawley rats were maintained on normal powdered diet or on the same diet containing 10 ppm chlordecone (CD) or 225 ppm phenobarbital (PB) for 15 days. On day 15, they received a single IP injection of a subtoxic dose of CCl4. Induction of cytochrome P-450 was greater with phenobarbital treatment than with chlordecone, but the CCl4-induced destruction of P-450 was similar in both groups and was progressive with the dose of CCl4 and with time after CCl4 administration. CCl4 given to animals on normal diet in a dose range of 25 to 200 microL/kg did not significantly alter the P-450 levels. These findings are consistent with greater bioactivation of CCl4 after the above two pretreatments There was a massive accumulation of Ca2+ in CD- and PB-pretreated animals after CCl4 administration, CD being more effective in this regard. Elevation of cytosolic Ca2+ was progressive despite the mitochondrial and microsomal sequestration of cytosolic Ca2+ at elevated levels. This perturbation of hepatocellular Ca2+ homeostasis which occurs 3 to 6 hr after CCl4 may prevent hepatocellular repair and renovation in CD-treated animals, leading to progressive hepatic lesion, hepatic failure and animal death by 36 to 48 hr at nontoxic doses of CCl4. Neither CD nor PB nor CCl4 alone affected hepatic Ca2+. These findings suggest that excessive Ca2+ accumulation may be related to the progression of hepatotoxic response to CCl4 in CD-treated animals.

The effects of route of exposure and combined exposure of mixed function oxidase inducers and suppressors on hepatic parameters in rainbow trout (Salmo gairdneri)

Hepatic microsomes obtained from rainbow trout ( Salmo gairdneri) administered β-naphthoflavone (βNF) either by a single intraperitoneally (IP) injected dose of 100 mg/kg body wt. or by feeding 500 ppm for 1 wk showed increased cytochrome(s) P-450 content and increased aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin- O-deethylase (ECOD) activities compared to controls. The microsomal maximum absorbance wavelength of carboxyferrocytochrome P-450 was 450 nm for the control trout, 448 nm for the trout administered βNF by diet, and 449 nm for the trout administered βNF by IP. Hepatic microsomes from trout fed 500 ppm phenobarbital (PB) for 3 wk showed no induction as demonstrated by cytochrome(s) P-450 content, NADPH-cytochrome c reductase, ethoxyresorufin- O-deethylase (EROD), ECOD, and benzphetamine- N-demethylase (BND) activities. The S20 liver fractions from trout fed βNF were less efficient in converting aflatoxin B 1 to a mutagen to Salmonella typhimurium TA 98 than were the S20 liver fractions from control trout. This suggests that dietary βNF may suppress the microsomal conversion of aflatoxin B 1 to the active 2,3-oxide in trout. Trout fed 500 ppm βNF for 3 wk followed by 3 more wk of βNF plus 0, 50, 150, 300, 450, or 600 ppm cyclopropenoid fatty acids (CPFA's) exhibited a significant decrease in the following parameters with an increase in dietary CPFA's: liver wt./body wt. percent, microsomal protein and cytochrome(s) P-450 content, and NADPH-cytochrome c reductase, ECOD, AHH, and glutathione transferase activities. Feeding 300 ppm CPFA's with 500 ppm βNF caused a shift in the maximum absorbance wavelength from 448 nm to 449 nm and decreased cytochrome(s) P-450 content by 71% whereas 450 ppm CPFA's caused a further shift to 450 nm and a corresponding 77% decrease in cytochrome(s) P-450 content. βNF induced and CPFA's suppressed the trout hepatic mixed function oxidase system.

Enhancing effect of butylated hydroxytoluene on the development of liver altered foci and neoplasms induced by N-2-fluorenylacetamide in rats

The enhancement of hepatocarcinogenesis by butylated hydroxytoluene (BHT) in comparison with that by phenobarbital (PB) was studied by quantifying their effects on N-2-fluorenylacetamide (FAA)-induced preneoplastic and neoplastic rat-liver lesions. Hepatocellular altered foci identified by iron exclusion and γ-glutamyltranspeptidase (GGT) activity were induced by feeding 0.02% FAA for 8 wk. Subsequently, BHT was fed at concentrations of 300, 1000, 3000 or 6000 ppm for up to 22 wk after cessation of FAA exposure; PB was fed at concentrations of 316 or 500 ppm for comparison. The lower doses of BHT (300, 1000 and 3000 ppm) did not exert a significant effect on either foci development or the final yield of neoplasms. At 6000 ppm, BHT increased the number of foci, the area occupied by GGT-positive preneoplastic and neoplastic lesions and the neoplasm incidence, as did 316 and 500 ppm PB. Comparison of the effects of BHT and PB at equimolar concentrations revealed that BHT was a much weaker enhancer of liver carcinogenesis. Apparently, the effective dose range of BHT as an enhancer is rather restricted. On the basis of available evidence that BHT is nongenotoxic and exerts epigenetic effects, we conclude that BHT is a weak promoter of liver carcinogenesis.

CCl 4-induced alterations in Ca ++ homeostasis in chlordecone and phenobarbital pretreated animals

Male S-D rats were maintained on normal powered diet or on the same diet containing 10 ppm chlordecone or 225 ppm phenobarbital for 15 days. On day 15, all the animals received a single ip injection of either corn oil or a subtoxic dose of CCl 4 (25–200 μl/kg) in corn oil vehicle (1 ml/kg). The animals were sacrificed 12 hrs later. Liver microsomal cytochrome P-450 and Ca ++ levels in whole liver, mitochondria, microsomes and cytosol were determined. Cytochrome P-450 induction was greater with phenobarbital pretreatment than with chlordecone but the CCl 4 induced destruction of cytochrome P-450 was almost similar in both groups and progressive with the dose of CCl 4. CCl 4 given to animals on normal diet in a dose range of 25–200 μ l/kg did not significantly alter the cytochrome P-450 levels. These findings are consistent with greater bioactivation of CCl 4 after the above two pretreatments. There was a massive accumulation of Ca ++ in chlordecone and phenobarbital pretreated animals after CCl 4 administration. Cytosolic Ca ++ levels remained high despite the mitochondrial and microsomal sequestration. This perturbation of hepatocellular Ca ++ homeostasis might lead to hepatic lesion and hepatic failure. Chlordecone or phenobarbital alone do not alter hepatic Ca ++ levels. These findings suggest that excessive accumulation of Ca ++ may be causally related to the progression of hepatotoxic response due to CCl 4 in chlordecone treated animals.

Tumor Promotion in Rat Liver

An initiation/promotion bioassay for chemical carcinogens and tumor promoters has been developed in rat liver using presumed preneoplastic lesions, foci of gamma-glutamyltranspeptidase (GGTase)-positive hepatocytes, as the endpoint. To evaluate the tumor-promoting activity of phenobarbital, rats were administered diethylnitrosamine (DENA), 2.0 mmole/kg, followed by 500 ppm phenobarbital in their drinking water. After 6 weeks of phenobarbital promotion, the rats had an increased incidence of foci as compared to nonphenobarbital-treated rats. By 50 weeks, the number of foci in the nonpromoted animals equaled the number observed with promotion. The stability and progression of GGTase-positive foci was determined in rats that received a 2/3 partial hepatectomy, followed 24 hours later by DENA administration (0.3 mmole/kg). The rats then received 500 ppm phenobarbital in the drinking water for 7 weeks. After 7 weeks, half of the rats were continued on phenobarbital and the other half were removed from phenobarbital treatment. The number of foci observed in rats continued on phenobarbital treatment leveled off after 10 weeks of promotion, while in rats taken off phenobarbital it did not regress but increased at a slower rate, and, by 56 weeks, approached the number observed in rats subjected to continuous promotion. At 56 weeks, the size of foci was larger after continuous promotion. At 81 weeks, all 6 (100%) of the rats on continuous promotion had liver tumors, while only 3 of 6 (50%) of the rats removed from promotion had tumors. Promotion by phenobarbital stimulated the growth and decreased the time required for the appearance of GGTase-positive foci and liver tumors.

Tumor promotion in rat liver.

An initiation/promotion bioassay for chemical carcinogens and tumor promoters has been developed in rat liver using presumed preneoplastic lesions, foci of gamma-glutamyltranspeptidase (GGTase)-positive hepatocytes, as the endpoint. To evaluate the tumor-promoting activity of phenobarbital, rats were administered diethylnitrosamine (DENA), 2.0 mmole/kg, followed by 500 ppm phenobarbital in their drinking water. After 6 weeks of phenobarbital promotion, the rats had an increased incidence of foci as compared to nonphenobarbital-treated rats. By 50 weeks, the number of foci in the nonpromoted animals equaled the number observed with promotion. The stability and progression of GGTase-positive foci was determined in rats that received a 2/3 partial hepatectomy, followed 24 hours later by DENA administration (0.3 mmole/kg). The rats then received 500 ppm phenobarbital in the drinking water for 7 weeks. After 7 weeks, half of the rats were continued on phenobarbital and the other half were removed from phenobarbital treatment. The number of foci observed in rats continued on phenobarbital treatment leveled off after 10 weeks of promotion, while in rats taken off phenobarbital it did not regress but increased at a slower rate, and, by 56 weeks, approached the number observed in rats subjected to continuous promotion. At 56 weeks, the size of foci was larger after continuous promotion. At 81 weeks, all 6 (100%) of the rats on continuous promotion had liver tumors, while only 3 of 6 (50%) of the rats removed from promotion had tumors. Promotion by phenobarbital stimulated the growth and decreased the time required for the appearance of GGTase-positive foci and liver tumors.

Dibromoethane effects on the induction of ?-glutamyl-transpeptidase positive foci in rat liver

The initiating and promoting activities of 1,2-dibromoethane in rat liver were investigated using the enzyme-altered foci bioassay. The incidence of γ-glutamyl-transpeptidase (GGT)-positive foci was used as an early histochemical marker for hepatocarcinogenesis. To determine the initiating activity of 1,2-dibromoethane, the halogenated hydrocarbon was administered orally in corn oil as single or multiple doses (60 or 120 mg/kg) either before or after partial hepatectomy. The animals were then given a promoting regimen of 500 ppm phenobarbital in their drinking water. No increase in the incidence of GGT-positive foci was observed in any of the 1,2-dibromoethane initiation groups. The tumor promoting activity of 1,2-dibromoethane was determined in partially hepatectomized rats which were initiated with N-nitrosodiethylamine (30 mg/kg; po), and one week later were administered 1,2-dibromoethane (10 or 30 mg/kg) orally in corn oil five times weekly for 8 weeks. Control groups receiving sham hepatectomy or no initiator were also treated with the halogenated hydrocarbon five times weekly. Only in those animals which received partial hepatectomy, N-nitrosodiethylamine initiation, and 1,2-dibromoethane was the incidence of GGT-positive foci significantly increased. These results do not support significant initiator activity of 1,2-dibromoethane in rat liver, but do indicate that 1,2-dibromoethane possesses promoter activity which may contribute to its carcinogenic activity.

Potentiation of CCl 4 lethality by chlordecone

Previous studies have dealt with the propensity of chlordecone (CD) to potentiate the hepatotoxicity of CCl 4. The effect of this interaction upon CCl 4 lethality has never been reported. The objective of the present study was to evaluate the effect of CD on CCl 4 lethality and several parameters associated with cytochrome P-450 activity. Male Sprague-Dawley rats (150–200 g) were fed powdered diets containing 0 or 10 ppm CD or 225 ppm phenobarbital (PB) for 15 days. On day 15, rats from each group received an i.p. injection of CCl 4 in corn oil and the 48-h LD50 was determined. Additional animals were used to measure hepatic microsomal levels of cytochrome P-450 and aminopyrine demethylase. CD decreased the LD50 of CCl 4 from 2.8 ml/kg to 0.042 ml/kg, representing a 67-fold increase in toxicity, as assessed by lethality. PB in this protocol decreased the LD50 to 1.7 ml/kg, representing a 1.6-fold increase in lethality. However, the induction of cytochrome P-450 and associated parameters of mixed-function oxidase (MFO) activity did not correlate with the lethality data. Hepatic microsomal P-450 was increased over controls by 40% and 76% in CD and PB rats respectively. PB also stimulated aminopyrine demethylase activity to a much greater extent (190%) than CD (49%) relative to controls. These findings provide evidence that the CD-CCl 4 interaction results in a marked increase in CCl 4 lethality, in addition to the previously reported marked increase in hepatotoxicity. These results remain consistent with the proposal that bioactivation is the mechanism underlying enhanced CCl 4 toxicity, but suggest that specific effects of CD upon CCl 4 metabolism may be the pivotal mechanism underlying potentiation.

Potentiation of brominated halomethane hepatotoxicity by chlordecone in the male rat

The potentiation of hepatotoxicity by CCl 4 and a series of brominated analogs by chlordecone (CD) and phenobarbital (PB) pretreatment was investigated, using individually nontoxic levels of the halomethanes and the potentiating agents. Hepatotoxicity was determined by biochemical, functional, and histopathological parameters. Male Sprague-Dawley rats were maintained on a diet of 0 or 10 ppm CD or 225 ppm PB for 15 days. On Day 15, the animals were challenged with an approximately equimolar ip dose (10 μl/kg) of CCl 4, CCl 3Br, CBr 4, or CHBr 3 in corn oil. Twenty-four hours later, biliary excretion of phenolphthalein glucuronide (PG) and bile flow were determined in intact cannulated animals over a 60-min period. While biliary excretion of PG was significantly decreased by CD-CCl 3Br and PB-CBr 4 combinations, the former combination was not accompanied by decreased bile flow. Of the four halomethanes tested, CD significantly elevated serum transaminase (SGPT, SGOT) and isocitrate dehydrogenase (ICD) activities only for the CCl 3Br combination, while phenobarbital treatment was without effect. Histopathological examination indicated that livers from the CD-CCl 3Br combination alone displayed extensive centrilobular necrosis. Based on these features of hepatotoxicity, CD resulted in readily observable potentiation of CCl 3Br hepatotoxicity. The PB-CCl 3Br combination did not result in potentiated CCl 3Br hepatotoxicity at the dose level utilized in this study. These results emphasize the extremely powerful nature of the CD-CCl 3Br interaction.

Comparative effect of pretreatment with phenobarbital, Aroclor 1254, and β-naphthoflavone on the metabolism of lindane

An attempt was made to distinguish different patterns of microsomal enzyme induction by phenobarbital, β-naphthoflavone, and Aroclor 1254 on the biotransformation and excretion of the organochlorine insecticide lindane. Treated groups of six weanling female Sprague-Dawley rats, individually housed in metabolism cages, received diets containing either 500 ppm Aroclor 1254, 356 ppm phenobarbital, or 418 ppm β-naphthoflavone. After 1 week all animals, except one group of controls, were dosed p.o. with 1.89 mg lindane (containing 1.63 μCi [U- 14C]lindane). Twenty-four hours later the rats were sacrificed and urine, feces, expired air, and tissue samples were taken for analysis of radioactivity. Hepatic cytochrome P-450 content, microsomal phospholipid content, and the enzyme activity involved in the dehydrogenation of lindane, the dechlorination of lindane, and the hydroxylation of the intermediate hexachlorocyclohexene were determined in vitro. Moreover, the effect of pretreatment on the excretion of radioactivity and the distribution of eight lindane metabolites was examined. Even though the rate of lindane metabolism was unchanged by the β-naphthoflavone pretreatment, results of the study indicated that all three pretreatments significantly altered lindane metabolism. The pretreatments differed from one another in that they selectively altered specific metabolic pathways.

Plasma alpha-fetoprotein elevation and mutagenicity of urine as early predictors of carcinogenecity in benzo(alpha)pyrene fed rats.

Weaning male Fischer rats were fed purified diets containing 0, 1, 100, or 1,000 ppm benzo(alpha)pyrene (BAP) for 6 weeks or 0, 1, or 100 ppm BAP for 3 weeks, then the same diets with or without 500 ppm sodium phenobarbital (PB) for an additional 5 weeks. Blood plasma alpha-fetoprotein (AFP) was determined at one to two week intervals. Urine, collected for eight hours during the sixth or eighth week of treatment, was tested for mutagenicity with S. typhimurium strain TA98 by the Salmonella/mammalian microsome test. Frozen liver sections were stained for gamma-glutamyl transpeptidase (GGT) activity. Only the 1000 ppm BAP treatment induced elevated blood plasma levels of AFP and GGT-positive hepatic cell foci. Urine from rats fed 1000 ppm BAP pr 100 ppm BAP + 500 ppm PB caused a significant (P less than or equal to .05) increase in revertants of S. typhimurium, compared to urine from control rats. Evidence from this investigation suggests that plasma AFP determination and urinary mutagenesis testing could be useful in short term in vivo screening for potential carcinogens.