Ploidy and specific karyotypic changes during promotion with phenobarbital, 2,5,2',5'-tetrachlorobiphenyl, and/or 3,4,3'4'-tetrachlorobiphenyl in rat liver.

Polychlorinated biphenyls are a group of industrial chemicals that are widely distributed in the environment. Since these compounds occur as mixtures, studies of their possible interactive effects are important. In order to determine whether an interaction of 2,5,2',5'-tetrachlorobiphenyl (TCB) with 3,4,3',4'-TCB occurs during multistage hepatocarcinogenesis in vivo, like that previously observed in lymphocytes in vitro (L. M. Sargent et al., Mutat. Res., 224: 79-88, 1989), we exposed rats to a single initiating dose of diethylnitrosamine (DEN), 10 mg/kg after a 70% partial hepatectomy, and subsequently to 0.1 ppm 3,4,3',4'-TCB and/or 10 ppm 2,5,2',5'-TCB in the diet for 1 year. Administration of each of the TCBs alone after DEN initiation resulted in a low incidence of chromosomal damage in hepatocytes; but when the two were given together after DEN initiation, there was a more than additive effect on this parameter at both 7 and 12 months which was highly significant. Administration of the TCBs alone or in combination in the absence of DEN initiation also resulted in chromosomal damage, approaching that seen in livers of animals initiated with DEN when sacrificed at 12 months. In animals receiving 0.05% phenobarbital for a 12-month period after initiation with DEN, a significant degree of chromosomal breakage and fragment formation occurred both in hepatocytes expressing the ectoenzyme gamma-glutamyltranspeptidase (GGT) and in those that were GGT negative. However, the GGT-negative cells showed a significantly lower incidence of chromosomal damage than the GGT-positive hepatocytes. Exposure to phenobarbital for 7 months after DEN initiation resulted in no significant chromosomal damage in hepatocytes, whether GGT positive or GGT negative. Some degree of specificity in chromosomal alterations was seen in hepatocytes of animals initiated with DEN and promoted either with a combination of TCBs or with phenobarbital. The most frequent alterations seen were a trisomy of chromosome 1 or of its long arm and a monosomy of chromosome 3 or its short arm. Some chromosome 7 aberrations were also seen. The highest frequency of specific aberrations occurred in hepatocytes from rats that also bore hepatocellular carcinomas, suggestive of the hypothesis that genes involved in the development of hepatic carcinoma may reside in chromosome 1 and/or 3 of the rat.     

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.     

Quantitative stereological evaluation of four histochemical markers of altered foci in multistage hepatocarcinogenesis in the rat

Female F344/N rats dosed with diethylnitrosamine (DEN) 24 hafter partial hepatectomy were treated with the promoting agents,phenobarbital (PB) or 3,4,7,8-tetrachlorodibenzo-p-dioxin (TCDD),or the peroxisome proliferating agent, WY 14,643, for 6 months.Another group was subjected to the Solt-Farber protocol. Alteredhepatic foci (AHF) were analyzed by quantitative stereologyfrom frozen serial sections stained for gamma-gtutamyl transferase(GGT), canalicular adenosine triphosphatase (ATPase), glucose-6-phosphatase(G6Pase) and the placental isozyme of glutathione S-transferase(PGST). PGST scored more foci in all groups than GGT and ATPase.PGST marked greater focal volume than GGT or ATPase, and PGSTmarked focal volume equal to or greater than G6Pase in ratstreated with PB, TCDD or the Solt-Farber protocol. However,after treatment with WY 14,643, GGT and PGST marked much lessfocal volume than ATPase or G6Pase, and PGST scored fewer focithan G6Pase. Numerical estimations of foci scored by those markerson the basis of area of the entire tissue section (per cm²)were relatively different from those values determined by quantitativestereology. While these results confirm earlier studies, theydemonstrate the importance of quantitative stereologic analysisof AHF during multistage hepatocarcinogenesis.     

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).     

Ovariectomy promotes the growth of altered hepatic foci after withdrawal and reintroduction of phenobarbital during hepatocarcinogenesis in rats.

Female F344/N rats were given 70% partial hepatectomies and intubated with diethyl-nitrosamine (DEN, 10 mg/kg) 24 hours later. They were fed a cereal-based diet, NIH-07 (NIH) + 0.05% phenobarbital (PB) for 6 months, at which time NIH + PB was withdrawn and the rats were ovariectomized (OV) or sham-operated (SH). Groups of 7-10 rats were fed a semipurified diet (AIN-76) for 1 or 2 months after withdrawal of NIH + PB, or NIH + PB for 2 months, or AIN-76 diet for 1 month and subsequently NIH + PB for 1 month. Placental glutathione S-transferase (PGST)- and gamma-glutamyltransferase (GGT)-positive (+) altered hepatic foci (AHF) were analysed by quantitative stereology. Ovariectomy stimulated growth of AHF after withdrawal and reintroduction of NIH + PB. AHF, especially PGST+ AHF, continued to regress throughout the PB withdrawal period in rats fed AIN-76 diet. In most studies of chemical hepatocarcinogenesis, females have been shown to develop a greater volume of AHF than males. In our study, however, ovariectomy stimulated the growth of AHF after withdrawal and reintroduction of PB. Because AHF occurring spontaneously in male rats develop more rapidly than in female rats, the greater rate of growth of AHF in OV female rats may reflect a similar mechanism.     

Focal and non-focal hepatic expression of placental glutathione S-transferase in carcinogen-treated rats

Carcinogenesis develops in stages that have been operationallydefined as initiation, promotion and progression. Although morphologicalend points have been described for detection and quantitationof these stages, to date initiation has been assessed only inthe context of clonal growth in response to certain promotingagents. Initiated cells are morphologicatly indistinguishablefrom surrounding cells and early changes at the cellular levelduring initiation have not been clarified. One commonly usedend point for the detection of preneoplastic hepatic lesionsis their aberrant expression of the placental isozyme of glutathioneS-transferase (PGST). Because single hepato cytes expressingPGST have been detected in aged rats and in those administeredhepatocarcinogens, it has been suggested that such cells constitutea population of putat ively initiated hepatocytes. In orderto further elucidate the characteristics of single PGST-positivehepatocytes, we analyzed the number of these cells 2 and 18weeks after various doses (0–100 mg/kg) of diethylnitrosamine(DEN) and of dimethylbenz[a]anthracene (DMBA). When determined14 days after carcinogen administration, the number of singlehepatocytes expressing PGST was greater after DEN administration(ranging from 0.8±0.3 per cm² transection of liver at1 mg/kg to 33.0±4.7 at 100 mg/kg) than after DMBA administration(ranging from 0.25±0.14 at 10 mg/kg to 3.03±0.5at 100 mg/kg); none were detected in control rats of the sameage. Additional rats were maintained on a basal diet or a basaldiet plus phenobarbital for a further 4 month period. Whereasindividual PGST-positive hepatocytes were only sporadicallydetected in rats treated with DMBA and maintained on a basaldiet for 18 weeks, those rats placed on phenobarbital for 16weeks had an even higher number of such PGST-positive hepatocytes than at 2 weeks after DMBA administration. In contrast,the dose-response curve observed for DEN- treated rats 18 weeksafter carcinogen administration was similar to that observed2 weeks after carcinogen treatment for both phenobarbital- andnon-phenobarbital-treated rats. In addition, the number of singlePGST-positive hepatocytes detected at 2 weeks was directly parallelto the number of altered hepatic foci expressing PGST 18 weeksafter DEN administration. The dose-dependent induction of PGST-positivesingle hepatocytes after treatment with two hepatocarcinogens,the dose-dependent growth of altered hepatic foci (AHF) expressingPGST with phenobarbital administration and the parallel dose-responsecurve of single hepatocytes expressing PGST and later of AHFexpressing PGST argue strongly for a precursor role of singlePGST-positive cells in the development of AHF expressing PGST.     

Altered growth control of rat hepatocytes after treatment with 3,4,3',4'-tetrachlorobiphenylin vivo and in vitro

Alterations of hepatocyte growth control by inducers of drugmetabolizingenzymes were investigated using 3,4,3',4'-tetrachlorobiphenyl(TCB) as the inducing agent. TCB was chosen as a selective 3-methylcholanthrene-typeinducer and liver tumor promoter which probably exerts its biologicalactions through binding to the aryl hydrocarbon (Ah) receptor.In vivo treatment of rats with TCB (200 mg/kg) markedly stimulatedgrowth of enzyme altered liver foci and [³H]-thymidine incorporationinto nuclear DNA. Hepatocyte cultures from TCB-treated ratswere more sensitive to exogenous growth factors such as EGFthan those from untreated controls. In vitro TCB exposure ofhepatocyte cultures also altered hepatocyte growth control ina dose-dependent manner and induced drug-metabolizing enzymes.TCB treatment in vivo enhanced EGF-stimulated autophosphorylationof the EGF receptor in liver plasma membranes. The results suggestthat altered growth control is due to a direct effect of TCBon hepatocytes. The proposed model may be useful to elucidatea possible linkage between the functional stress imposed onhepatocytes by sustained overexpression of an adaptive programand modulation of hepatocyte growth control.     

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.     

Sequential study on the synergistic effects of 2,2',4,4',5,5'-hexabromobiphenyl and 3,3',4,4',5,5'-hexabromobiphenyl on hepatic tumor promotion

A sequential study was completed to determine the effect ofpolybrominated biphenyl (PBB) congeners on the enhancement ofgamma-glutamyl transpeptidase (GGT)-positive altered hepaticfoci (AHF) and the development of hepatic nodules (HN) and carcinomas.Female Sprague-Dawiey rats were given a single dose of N-nitrosodiethylamine(NDEA) 24 h following a 70%partial hepatectomy. Thirty dayslater, rats were randomly assigned to groups and fed a basaldiet or the basal diet containing 10 p.p.m. 2, 2', 4, 4', 5,5'-hexa-bromobiphenyl (245-HBB), 0.1 p.p.m. 3, 3', 4, 4', 5,5-hexa-bromobiphenyl (345-HBB) or 10 p.p.m. 245-HBB plus 0.1p.p.m. 345-HBB for 140 days followed by a basal diet for upto another 310 days. Rats from each group were killed 170, 240or 480 days after partial hepatectomy. Dietary exposure to 245-HBBand 245-HBB plus 345-HBB enhanced the development of AHF andHN whereas 345-HBB alone did not. The combination of 245-HBBand 345-HBB caused a synergistk effect on the development ofAHF and HN. The number of hepatocellular carcinomas was lowand evenly distributed among the groups of rats fed diets containingPBB.     

Quantitative stereologic study of the effects of varying the time between initiation and promotion on four histochemical markers in rat liver during hepatocarcinogenesis

The effects of varying the interval of time between initiation with diethylnitrosamine (DEN) and promotion by phenobarbital (PB) on the development of altered hepatic foci (AHF) and hepatomas in female Fischer 344 rats was investigated. The intervals between DEN initiation after a 70% partial hepatectomy and a subsequent 6 month period of promotion by feeding of PB were 1 day, 1 week, 1 month, 2 months, 6 months and 11 months. The number and volume percentage occupied by AHF were determined by quantitative stereologic methods on serial frozen sections stained for the markers gamma-glutamyltranspeptidase (GGT), canalicular adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6Pase) and the placental form of glutathione S-transferase (GST-pi). The number of AHF was greatest when the initiation-promotion interval was only 1 day, and there was a tendency for the number of AHF to decrease as the interval between initiation with DEN and the start of PB promotion was extended. An 11 month delay between initiation and promotion resulted in only 20% fewer AHF than when promotion was begun 1 day after initiation. On the other hand, the volume percentage fraction of AHF did not change when the initiation-promotion interval was increased from 1 day to 2 months. An interval of 6 months roughly doubled the volume percentage fraction, but an interval of 11 months led to a 7- to 8-fold increase in the volume percentage of AHF over that from a 1 day interval. The phenotypic distribution of AHF was significantly lower in relation to certain markers, especially GGT and GST-pi, in those animals only initiated with DEN compared with those initiated with DEN and promoted with PB. When no exogenous promotion was given, there was still a nearly linear increase in both the number and volume percentage occupied by AHF in the liver of rats initiated with DEN. On the other hand, rats subjected to a 1 week interval between DEN initiation and PB promotion exhibited the greatest number of hepatocellular carcinomas 14 months after initiation, compared with other groups. These studies demonstrated a gradually decreasing effectiveness of PB as a promoting agent to stimulate the growth of all AHF initiated by DEN as the interval between initiation and promotion was extended.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of choline and methionine deficiencies on the number and volume percentage of altered hepatic foci in the presence or absence of diethylnitrosamine initiation in rat liver

The ability of methyl-deficient, amino-acid-defined diets to produce enzyme-altered foci was quantitatively determined in the livers of rats treated both with and without an initiating dose of diethylnitrosamine (DEN). Male weanling F-344 rats were fed a complete, amino-acid-defined diet for 1 week. They were then injected i.p. with a single dose of DEN (20 mg/kg body weight) and fed the complete diet for an additional week. Forty animals in each dose group were then maintained for 5-38 weeks on the complete diet (diet 1) or one of the three methyl-deficient diets customarily used in this laboratory: diet 2, devoid of methionine and choline; diet 3, devoid of methionine only; and diet 4, devoid of choline only. In diets 2 and 3, methionine was replaced by equimolar amounts of its metabolic precursor, DL-homocystine. Ten animals per group were killed 8, 12, 17, 24 and 41 weeks after DEN initiation. For 2 weeks prior to being killed, each group was maintained on the complete diet to minimize the histological abnormalities due to acute toxicity of the diets. Serial sections of the livers were obtained, stained sequentially for gamma-glutamyltranspeptidase, ATPase and glucose-6-phosphatase, and the quantitation of the focal lesions scored by these markers was carried out by quantitative stereology. The results indicated that, regardless of the enzyme marker(s) examined, there was a general correspondence between the volume and number of altered hepatic foci (AHF) formed and the previously described tumor-promoting activities of each diet. Thus, while all DEN-treated groups contained significant numbers of AHF 24 weeks after initiation, only the diet-2-fed animals displayed such foci at 8 weeks. Similarly, among the uninitiated rats, only those fed diet 2 exhibited the presence of AHF throughout the experimental period. Interestingly, the livers of uninitiated, choline-deficient rats showed a small number of AHF at 24 and 42 weeks; these foci were not observed at all in the corresponding DEN-untreated animals fed diet 3, deficient in methionine only. The results provide evidence that the carcinogenic effects of the methionine- and choline-deficient diet result more from its strongly promoting effect than from any initiating activity by the diet.     

Modulation of altered hepatic foci induction by diallyl sulphide in Wistar rats.

Diallyl sulphide (DAS) is a sulphur-containing volatile compound present in garlic (Allium sativum). It has been shown to inhibit a number of chemically induced forms of cancer in experimental animals. The present study demonstrates the inhibitory effect of DAS on the development of diethylnitrosamine (DEN) initiated and 2-acetyl-aminofluorene (2-AAF) promoted preneoplastic altered hepatic foci (AHF) in Wistar rats. AHF were scored and analysed by quantitative stereology using the Image Analysis system from frozen liver sections stained for biological markers, namely glutathione S-transferase, placental form (GST-P), gamma-glutamyl transpeptidase (GGT), adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6 Pase) and alkaline phosphatase (AlkPase). DAS-supplemented rats were found to restore the near-normal levels of enzymes GST-P and GGT when exposed to DEN and 2-AAF. DAS administration following DEN and 2-AAF exposure led to the restoration of enzymic activity of ATPase, G6 Pase and AlkPase, as evident by number and area of the foci. These findings suggest the protective role of DAS in rat hepatocarcinogenesis, by suppressing DEN- and 2-AAF-induced AHF development.     

Alteration in expression of gap junction proteins in rat liver after treatment with the tumour promoter 3,4,5,3',4'-pentachiorobiphenyl

Polychiorinated biphenyls (PCBs) are industrial chemicals whichare highly persistent and widely distributed in the environment.We have previously shown that 3,4,5,3',4'-pentachiorobiphenyl(PCB 126) is a potent tumour promoter in two separate 20 weekinitiation–promotion studies. In the present study, ratlivers from these two studies were further investigated forconnexin expression. The results demonstrated that treatmentwith PCB 126 caused a decrease in the amount of the two majorliver connexins, cx 26 and cx 32, In livers of treated animals.This reduction was also prominent after treatment at low doses,although     

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.     

Quantitative Comparison of Initiation and Mutation Phenotypes in Hepatocytes of the Analbuminemic Rat

The potential relationship between mutagenesis and carcinogenesis has been examined in the Nagase analbuminemic rat treated with a single dose of benzo[a]pyrene, an incomplete liver carcinogen. The apparent mutation rate at the albumin locus was calculated by determining the number of hepatocytes expressing a cross-reactive product of albumin in analbuminemic rats treated with benzo[a]pyrene. The rate of initiation, the first stage in carcinogenesis, was determined by assessing the number of hepatocytes expressing the placental isozyme of glutathione S-transferase (PGST) after administration of benzo[a]pyrene. Since the expression of PGST may represent hepatocellular changes independent of initiation, promotion with phenobarbital was employed to clonally expand those putatively initiated hepatocytes expressing PGST. With immunohistochemical measures to assess changes in albumin expression, a threefold increase in the number of hepatocytes expressing albumin was detected after administration of benzo[a]pyrene in Nagase analbuminemic rats. A more than five-fold increase in altered hepatic foci (AHF) exhibiting increased PGST expression was observed in animals given benzo[a]pyrene treatment followed by phenobarbital, compared with those given benzo[a]pyrene alone. The number of albumin-expressing single hepatocytes detected was of the same order of magnitude as the number of individual hepatocytes and AHF expressing PGST, suggesting that similar events may be involved in their formation. Since 3 × 10⁶ single hepatocytes expressing albumin were found in the analbuminemic rat liver after a single administration of benzo[a]pyrene, while less than 2 × 10⁴ AHF expressing PGST were observed, formation of individual hepatocytes expressing albumin was a far more frequent event than clonal expansion of initiated hepatocytes in the Nagase analbuminemic rat. However, the number of loci of PGST expression including AHF and single hepatocytes is comparable to that of single hepatocytes expressing albumin.     

Effects of a single dose of polychlorinated biphenyls to infant mice on N-nitrosodimethylamine-initiated lung and liver tumors

Polychlorinated biphenyls (PCBs) are widespread, chemically-stable environmental contaminants; some congeners are commonly found in human adipose tissue and breast milk. We investigated the effects of a single dose of one PCB mixture (Aroclor 1254) on tumors initiated by N-nitrosodimethylamine (NDMA), also a common environmental agent. Infant outbred Swiss male mice were treated with NDMA (5 mg/kg) i.p. on the 4th day of life, to initiate lung and liver tumors. Four days later each received a single intragastric dose of PCBs (50, 250, or 500 mg/kg of Aroclor 1254) or oil. Groups were killed 16 and 28 weeks later. At both endpoints the mice given 500 mg/kg PCBs after NDMA developed twice as many lung tumors (alveologenic adenomas) as those treated with NDMA only, a significant difference. The PCBs alone did not cause lung tumors. This is the first demonstration of tumor promotion by PCBs in an extrahepatic organ, and it occurred after a single exposure. There were also complex, multiple effects on NDMA-caused liver tumors (adenomas and carcinomas) and on focal hepatocellular proliferative lesions: PCB treatment after the NDMA was associated with decreased number but increased size of these tumors and foci. All of these changes were accompanied by retention in the bodies of 0.1-6 ppm PCBs, as indicated by gas chromatography with electron capture detection. Of this, 80% or more consisted of 2,4,5,2',4',5'-and 2,3,4,2',4',5'-hexachlorobiphenyls in about equal amounts for periods up to 28 weeks. These results point to a need for both experimental and epidemiological studies of the effect of PCB body burden on tumor development.     

Expression of c-myc in altered hepatic foci induced in rats by various single doses of diethylnitrosamine and promotion by 0.05% phenobarbital

Among the proto-oncogenes examined by northern blot analysis, c-myc, c-Ha-ras, c-fos, and c-raf-1 have been reported to be activated in rat liver cell carcinomas. However, there are relatively few reports on proto-oncogene expression in altered hepatic foci (a) early during hepatocarcinogenesis in the rat. In this study, diethylnitrosamine (a) at doses ranging from 10 to 200 mg/kg was used to initiate and phenobarbital (0.05%) to promote AHF in rats. AHF were detected by the presence of the marker enzymes glutathione s-transferase, placental form (GST-P); γ-glutamyltranspeptidase (a); glucose-6-phosphatase (G6Pase); and canalicular ad-enosine triphosphatase (a). Proto-oncogene expression in individual AHF was investigated by in situ hybridization (a). ISH for the mRNAs of c-Ha-ras, c-fos, and c-raf-1 revealed little or no expression in AHF. However, the levels of c-myc mRNA were increased in about 10% of the AHF initiated by the highest dose of DEN (200 mg/kg). Thus, altered expression of proto-oncogenes was not seen in AHF initiated by nonnecrogenic doses of DEN and promoted by phenobarbital. However, at the necrogenic dose of 200 mg/kg DEN, c-myc expression was found mostly in AHF in which abnormal expression of GST-P, GGT, G6Pase, and ATPase was also present, indicating that c-myc expression is correlated with phenotypically greater complexity of the AHF, a characteristic of malignant hepatic neoplasms in the rat. © 1995 Wiley- Liss, Inc.     

The effect of the dose of diethylnitrosamine on the initiation of altered hepatic foci in neonatal female rats

The dose—response characteristics of initiation of hepatocarcinogenesisby diethylnitrosamine (DEN) was investigated in the neonatalfemale rat by means of the quantitative stereologic estimationof altered hepatic foci (AHF) expressing multiple markers. At5 days of age, female Sprague—Dawley rats were given asingle i.p. dose of DEN (0.1–30 mg/kg body wt) or thevehicle (trioctanoin). The semisynthetic AIN-76A diet was providedto half of the rats in each treatment group, while the remainderreceived this diet containing 500 mg phenobarbital (PB)/kg for8 months from weaning until the animals were killed. To ascertainmore exactly the dose—response relationship for initiationby DEN, the number, volume percentage and phenotypes of theresulting AHF were determined by quantitative stereologicalanalysis on serial sections of frozen tissue, each stained forone of four markers of preneoplasia. A linear relationship wasobserved between the dose of DEN (0–30 mg/kg) and thenumber and volume percentage of AHF detected, with each singlemarker or the total number of AHF detected when the placentalisozyme of glutathione S transferase,     

Enhancing effect of co-administration of polychlorinated biphenyls and diethylnitrosamine on enzyme-altered islands induced by diethylnitrosamine in rat liver

The effect of co-adminstration of diethytnitrosamine (DEN) andClophen A 50, a commercial mixture of polychlorinated biphenyls(PCB), on pre-neoplastk enzyme-altered islands in livers offemale Sprague-Dawley rats was studied. The islands were identifiedby the loss of adenosine-5'triphos-phatase (ATPase), emergenceof gamma-glutamyltranspep-tidase (GGTase) and glycogen storageafter fasting. DEN was given p.o. (0.4 or 4 mg/kg body wt respectively)twice a week for 11 consecutive weeks. Clophen A 50 (1 or 5mg/kg body wt respectively) was given alternatively three timesa week for 11 weeks. Four groups of rats each received eitherDEN or PCBs in the respective doses. Control animals were treatedwith the vehicle or remained untreated. All animals were killedat week 12. In rats treated with 4 mg DEN/kg body wt 80 ATPase-defidentislands/cm2 were observed. Additional treatment with ClophenA 50 enhanced the island number 3-fold. Treatment with 0.4mg/kg body wt DEN induced 17 islands/cm². Additional applicationof Clophen A 50 enhanced the island number 3-fold. The totalisland area was enhanced to the same extent in both groups.The island incidence in PCB-treated rats and controls was belowI/cm² with all markers tested. The results indicate that PCBsmay exhibit a co-carcinogenic activity.