Dose dependent induction of DNA adducts, gene mutations, and cell proliferation by the antiandrogenic drug cyproterone acetate in rat liver.

1. CPA does not only induce the formation of DNA adducts but also of mutations in female rat liver. 2. The mutation frequency exhibited a characteristic time course. Within a period of 3 days post administration, a tremendous increase was noted, which remained at a high level until 2 weeks post exposure. Thereafter, most mutation-carrying cells were eliminated within a period of 2 weeks leaving a cell population remaining at a constant level for another 4 weeks. Thus, the length of the observation period post exposure, i. e. the manifestation time, seems to be a critical factor for the strength of the mutagenic response. The highest as observed between 1 and 2 weeks post exposure. Correspondingly, the dose response curve recorded 2 weeks post exposure showed a higher mutagenic response than the curve after 6 weeks of exposure recorded previously. 3. When CPA-induced mutations were recorded as a function of the dose, mutation frequencies at the lower dose range were found that did not differ from those of controls. The non-effective dose recorded 2 weeks post exposure was much lower than that recorded after 6 weeks of exposure indicating that it is a function of the manifestation time. Since DNA adducts were formed in high amounts at the non-effective doses, we assume that the mitogenic activity required for the conversion of DNA adducts into mutations was not sufficiently strong. The liver of adult animals exhibits a very low endogeneous proliferation rate, which is not likely to contribute significantly to the expression of mutations. We conclude that it is the mitogenic activity of CPA itself, which stimulates the expression of mutations.

The DNA damaging drug cyproterone acetate causes gene mutations and induces glutathione-S-transferase P in the liver of female Big Blue transgenic F344 rats.

The gestagenic and antiandrogenic drug cyproterone acetate (CPA) is mitogenic, tumorigenic and induces DNA-adducts and DNA-repair synthesis in rat liver. Thus CPA is expected to be mutagenic. However in vitro mutagenicity test systems were negative. To examine whether CPA induces mutations in rat liver, the in vivo mutation assay based on Big Blue transgenic F344 rats was employed. Single oral doses of 25, 50, 75, 100 and 200 mg CPA/kg b.w. respectively were administered to female Big Blue rats. Six weeks after treatment, liver DNA was assayed for mutations. At the highest dose, 200 mg CPA/kg b.w., the frequency of (17 +/- 4) x 10(-6) spontaneous mutations was increased to a maximum of (80 +/- 8) x 10(-6) mutations. One-hundred and 75 mg CPA/kg b.w. resulted in mutation frequencies of (35 +/- 5) and (27 +/- 5) x 10(-6), respectively. The mutation frequency at doses of 50 and 25 mg CPA/kg b.w. was similar to that of vehicle treated controls. Statistical analysis of the dose-effect relationship revealed that it was not possible to decide whether a threshold dose exists or not. DNA adducts were analyzed by the 32P-postlabelling technique. The total level of the major and the two minor adducts observed in the autoradiograms increased between doses of 25 to 75 mg CPA/kg b.w. to a maximum of approximately 12,000 +/- 3000 adducts per 10(9) nucleotides. The level did not further increase significantly with 100 and 200 mg CPA/kg b.w. After CPA treatment no preneoplastic liver foci were observed. However, single glutathione-S-transferase placental form (GST-P) positive hepatocytes were observed and the frequency was dependent on the dose. These cells are not supposed to represent initiated cells, since they occurred only transiently after 6 weeks and disappeared thereafter completely. In conclusion, our results demonstrate that CPA is mutagenic in vivo. The mutation frequency increased at high CPA doses, when the increase of the DNA adduct formation had already ceased. This suggests that the mitogenic activity of CPA is required to express the mutations.

Chlorinated dibenzodioxins and dibenzofurans (PCDD/F) in blood and human milk of non occupationally exposed persons living in the vicinity of a municipal waste incinerator.

The concentrations of chlorinated dibenzodioxins and dibenzofurans (PCDD/F) in human blood and in milk from non-occupationally exposed persons living in the vicinity of a municipal waste incinerator were determined. As compared to background levels in the general population in Germany the results give no indication of an enhanced body burden of PCDD/F. This is in agreement with earlier investigations in the same area, showing normal background concentrations in soil, fruit and vegetables. In conclusion, no direct health hazard related to PCDD/F-emissions from a local municipal waste incinerator may be expected.

C-raf expression in early rat liver tumorigenesis after promotion with polychlorinated biphenyls or phenobarbital.

1. The expression of c-raf protooncogene in early stages of chemically induced rat liver tumorigenesis was studied in weanling female and adult male Sprague-Dawley rats. After initiation with diethylnitrosamine, promotion by polychlorinated biphenyls (PCBs) or phenobarbital (PB) was studied in the female. Male rats were promoted with PCBs only. 2. The incidence of enzyme-altered foci was evaluated histochemically by demonstrating a deficiency in adenosine-5'-triphosphatase and the emergence of gamma-glutamyl-transpeptidase. C-raf expression was measured in liver tissue containing preneoplastic foci, and in small (< 3 mm in diameter) and large (> 3 mm in diameter) neoplastic nodules up to 36 weeks. 3. Foci numbers amounted to 60-70 per cm2 liver section with both histochemical markers and both promoters in female rats. In male rats foci numbers were about 20-40 per cm2 liver section with both markers and with PCBs as promoting agents. Foci area developed more rapidly in female rats. 4. Small and large nodules were found in females during the entire observation period with both promoting agents, PCBs being more effective than PB. C-raf expression in nodules was increased up to 10-fold in PCB-treated animals compared with untreated controls. No dependence on the size of the nodules was seen. In male rats nodule incidence was very low and c-raf induction was marginal. 5. In conclusion, c-raf proto-oncogene expression correlated with the incidence of foci and nodules, female rats being more sensitive than males.

Initiation of enzyme-altered foci by the synthetic steroid cyproterone acetate in rat liver foci bioassay.

Cyproterone acetate (CPA) is a synthetic steroid which is used in oral contraceptive and anti-androgen formulations. It has previously been classified as a tumor promoter in rat liver. Recent studies have shown that CPA induces DNA repair synthesis in isolated hepatocytes, and this implies that CPA is genotoxic. We studied the initiating activity of CPA in vivo by means of a rat liver foci bioassay, using weanling female Sprague-Dawley rats. The results show that CPA induces adenosine-triphosphatase-deficient and gamma-glutamyltranspeptidase-positive putative preneoplastic foci in a dose-dependent manner. This indicates that CPA has not only promoting but also initiating activity and may therefore act as a complete carcinogen in rat liver.

Di(2-ethylhexyl)phthalate alters carbohydrate enzyme activities and foci incidence in rat liver.

The effect of di(2-ethylhexyl)phthalate (DEHP) on diethylnitrosamine (DEN)-initiated preneoplastic liver lesions with expression of gamma-glutamyltranspeptidase (GGTase) and loss of adenosine triphosphatase (ATPase) as well as alterations of hepatic carbohydrate metabolism in male and female Sprague-Dawley rats have been investigated. Two treatment schedules have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic islands and by the biochemical determination of alterations in enzyme activities of liver homogenates and of serum, the last indicating hepatotoxicity. For initiation, a single dose of DEN was given, followed by treatment with various doses of DEHP given three times weekly by gavage for 7 or 11 consecutive weeks. As histochemical enzyme markers, the expression of positive GGTase as well as the deficiency in ATPase were used for identification of liver foci. The weanling female rats (protocol A) were found to be more sensitive to the carcinogenic effect of DEN in view of foci incidence than the mature male rats which underwent partial hepatectomy prior to DEN application. The administration of 200 mg DEHP/kg body wt increased the incidence of ATPase-deficient foci in both male and female rats; however, concentrations of 1000 and 2000 mg DEHP/kg decreased the incidence of liver foci. The number of foci with expression of GGTase was only slightly increased in female rats following a DEHP concentration of 50 mg/kg, and 200 mg/kg body wt. DEHP alone did not induce preneoplastic lesions that could be identified by these two markers. Biochemical investigations indicate that DEHP alters the metabolic pattern in liver. An increase of the NADP-linked enzymes glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme, extra-mitochondrial ICDH as well as an enhancement of NAD-dependent alpha-G3PDH and lactate dehydrogenase were found following DEHP administration. On the other hand the glycolytic enzymes pyruvate kinase (PK) and enolase as well as the gluconeogenetic enzyme fructose-1,6-bisphosphatase (FBPase) were significantly reduced. In protocol B (male rats) the reactions of PK, FBPase and malic enzyme were more altered after DEHP exposure than in protocol A, while the activity of G6PDH was more increased in protocol A. Most enzymes being involved in the carbohydrate metabolism are influenced by DEHP in a dose-dependent manner. There was no increase in serum FBPase activity in both male and female rats after DEHP treatment but a reduction of glutamate-oxalate-transaminase and glutamate-pyruvate-transaminase activities was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Dose-dependent emergence of preneoplastic foci in rat livers after exposure to 2-nitropropane.

Male and female Sprague-Dawley rats, 4-6 days old were exposed for 3 weeks (6 h/day, 5 days/week) to 2-nitropropane vapours of 0, 25, 40, 50, 80 and 125 ppm. One week later polychlorinated biphenyls (Clophen A50, 10 mg/kg body weight) were administered for promotion twice a week for 8 weeks. Thirteen weeks after starting the experiments the logarithms of the numbers of preneoplastic liver foci deficient in adenosine-5'-triphosphatase were found to be linearly related to the exposure concentrations of 2-nitropropane. Male rats exhibited an approximately four times lower foci incidence than females.

Detection of chemical carcinogens by means of the "rat liver foci bioassay".

The rat liver foci bioassay designed as short-term screening test system in vivo is a reliable procedure for the detection of the carcinogenic potential of chemicals. The use of tumor prestages as markers is favorable in many respects: 1. There is a convincing correlation between foci and tumors. 2. The sensitivity is by a factor of more than 1,000 higher compared to the chronic carcinogenicity study. 3. It is the only system available so far for detecting liver tumor promotors. 4. There is promising prospect that carcinogenic agents with extrahepatic target organs can be detected.

Biological activity of 2,4,8-trichlorodibenzofuran: promotion of rat liver foci and induction of cytochrome P-450-dependent monooxygenases.

The biological activity of 2,4,8-trichlorodibenzofuran (2,4,8-TCDF) was tested using 2 endpoints: (a) the promotion of enzyme-altered, preneoplastic foci initiated by diethylnitrosamine (DEN) in livers of weanling female Sprague-Dawley rats; and (b) the induction of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), a marker for cytochrome P-4501 activity, in livers of adult female Sprague-Dawley rats and in H4IIEC3 rat hepatoma cells. When animals were treated with 200 or 500 mg/kg 2,4,8-TCDF 5 X weekly over 10 weeks after a single application of 10 mg/kg DNA, the higher dose of 2,4,8-TCDF had a promoting effect on the appearance of preneoplastic foci. Thus number and total area of foci deficient in adenosine-5'-triphosphatase were significantly increased by a factor of 1.6. 2,4,8-TCDF induced AHH-activities in 9000 X g supernatants of liver 2-3-fold, when rats were treated with 100-1000 mg/kg/day for 5 days and monooxygenase activities determined after another 3 days. The amounts of 2,4,8-TCDF required for inducing AHH activity in H4IIEC3 cells were 7 orders of magnitude higher than those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). the results indicate that the 2,4,8-TCDF has a biological activity which is extremely low compared to that of 2,3,7,8-TCDD.

Comparison of three rat liver foci bioassays--incidence of preneoplastic foci initiated by diethylnitrosamine.

Three rat liver foci bioassays have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic foci, and by the biochemical determination of alterations in enzyme activities of serum indicating hepatotoxicity. We studied the initiation/promotion schedules according to Oesterle and Deml (A), and according to Pereira (B, Broad Spectrum Protocol), and the initiation/selection protocol according to Tatematsu et al. (C), with diethylnitrosamine (DEN), given as a single initiating dose of 10 and 30 mg/kg body wt respectively. With all schedules Sprague-Dawley rats, either females, 3 weeks old (A), or males, 6 weeks old (B, C) were used. For promotion polychlorinated biphenyls (A) or phenobarbital (B) were administered. Selection was performed with 2-acetylaminofluorene (C). The rats in schemes (B) and (C) underwent partial hepatectomy one day prior to initiation. The number and total area of foci deficient in adenosine-5'-triphosphatase (ATPase) and positive in gamma-glutamyltranspeptidase (GGTase) was evaluated. In the complete schedule with 30 mg of DEN in system (A) foci incidence exceeded that of the other systems by about 7-fold (ATPase) and 2-fold (GGTase) respectively. The lower dose of DEN and all control experiments resulted in a respective lower foci yield. With scheme (C), but not with schemes (A) and (B), e.g. serum fructose-1.6-bisphosphatase and alkaline phosphatase were increased, suggesting liver cell damage. Thus tested with DEN, scheme (A) is most sensitive and causes a low impairment of animals' welfare.

Lack of initiating and promoting activity of thiourea in rat liver foci bioassay.

Thiourea (TU) fails to enhance the incidence of foci deficient in adenosine-5'-triphosphatase (ATPase) either by initiation or by promotion in a rat liver foci bioassay. To weanling female Sprague-Dawley rats, 3 x 200 or 3 x 500 mg/kg body wt of TU, respectively, were applied for initiation. One week later Clophen A 50, a technical mixture of polychlorinated biphenyls (PCBs) 2 x 10 mg/kg body wt were given twice weekly as promoting agent for 11 weeks. For promotion 0.2% of TU was administered with the drinking water for 51 days to rats of both sexes, and 0.1% and 0.05% of TU, respectively, for 70 days to females after initiation with 1 x 8 mg/kg body wt of diethylnitrosamine (DEN).

Inhaled ethylene oxide induces preneoplastic foci in rat liver.

The metabolite of E, EO, has been shown to be an extrahepatic carcinogen in rats in long-term studies. By means of a rat liver foci bioassay with 3 to 4 days old Sprague-Dawley rats, EO showed an initiating capacity in the livers of female, but not of male rats, measured as incidence of foci deficient in ATPase. After inhalation of 55 and 100 ppm EO, 8 h daily, 5 days weekly, and over 3 weeks, 1 week of pause, and another 8 weeks of promotion with polychlorinated biphenyls, foci incidence was generally low. But it was concentration dependently higher than in controls 12 weeks after starting the experiment. A linear concentration-effect relationship existed with a correlation coefficient of r = 0.991. With 33 ppm EO the number of foci was not enhanced significantly. The administration of 10,000 ppm E did not result in an enhanced foci incidence. In general the carcinogenic potential of EO, which has not been shown so far to cause hepatic tumors in rats, could be demonstrated in rat liver using a sensitive rat liver foci bioassay.

Promoting activity of di(2-ethylhexyl)phthalate in rat liver foci bioassay.

The plasticizer DEHP but not DEHS exerted a weak promoting effect in a 12-week rat liver foci bioassay, using weanling female Sprague-Dawley rats. The effect was similar after doses of 200 and 500 mg/kg body weight, given 3 times weekly by gavage for 11 consecutive weeks after initiation with a single oral dose of 8 mg DEN/kg body weight. Lower doses were ineffective. The incidence of foci with deficiency in ATPase was enhanced about twice compared to rats treated with DEN only. The incidence of foci with expression of GGTase was not affected by DEHP treatment. The results match the findings with lifetime exposure studies, when liver tumors were found in rats and mice. The actual risk for man from environmental DEHP contamination seems to be low; the intake from highly contaminated food is calculated to be about 400-fold lower than the lowest effective dose in this study.

Segmentation of scenes in tissue sections.

The segmentation of scenes of fixed tissue sections for quantitative histopathology is the crucial step for further image processing. Different segmentation methods for the separation of nuclei, nucleoli and whole cells in methacrylate-embedded sections of rat liver were investigated. Reasonable segmentation results were obtained using a contrast-enhanced polar-coordinate transformation to distinguish nuclei, a compactness algorithm to distinguish nucleoli and a skeletonization algorithm to delineate cells when a priori information on the morphometric and photometric properties of the liver tissue was included.

Dose-response of promotion by polychlorinated biphenyls and chloroform in rat liver foci bioassay.

Using the rat liver foci bioassay a dose-response relationship was evaluated for the promoting activity of the ubiquitous and persistent environmental pollutants polychlorinated biphenyls (PCBs) and chloroform. Initiation of liver foci was performed by oral administration of a single dose of 8 mg/kg body wt of diethylnitrosamine to weanling female Sprague-Dawley rats. For polychlorinated biphenyls (PCBs) and chloroform a dose-dependent promoting effect was found. The lowest effective dose of PCBs was 1 mg/kg body wt, given three times a week for 11 consecutive weeks. That of chloroform was 100 mg/kg body wt, administered twice a week for 11 consecutive weeks. The livers were screened for preneoplastic foci 12 weeks after starting the experiments. The amounts of PCBs as well as chloroform normally taken up by humans are greater than a factor of 1000 lower than the effective experimental doses. Thus the risk of human exposure to these chemicals is estimated to be very low. In the case of heavy pollution with PCBs, however, as happened in the yusho accident (Japan, 1968), the daily intake of PCBs was in the range of the effective doses used in the experiment.

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

The effect of co-administration of diethylnitrosamine (DEN) and Clophen A 50, a commercial mixture of polychlorinated biphenyls (PCB), on pre-neoplastic enzyme-altered islands in livers of female Sprague-Dawley rats was studied. The islands were identified by the loss of adenosine-5'-triphosphatase (ATPase), emergence of gamma-glutamyltranspeptidase (GGTase) and glycogen storage after 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 5 mg/kg body wt respectively) was given alternatively three times a week for 11 weeks. Four groups of rats each received either DEN or PCBs in the respective doses. Control animals were treated with the vehicle or remained untreated. All animals were killed at week 12. In rats treated with 4 mg DEN/kg body wt approximately 80 ATPase-deficient islands/cm2 were observed. Additional treatment with Clophen A 50 enhanced the island number 3-fold. Treatment with 0.4 mg/kg body wt DEN induced 17 islands/cm2. Additional application of Clophen A 50 enhanced the island number approximately 3-fold. The total island area was enhanced to the same extent in both groups. The island incidence in PCB-treated rats and controls was below 1/cm2 with all markers tested. The results indicate that PCBs may exhibit a co-carcinogenic activity.

Dose-dependent promoting activity of chloroform in rat liver foci bioassay.

Chloroform enhances dose-dependently the number of preneoplastic foci in livers of weanling female Sprague-Dawley rats. The preneoplastic foci were induced with a single dose of 8 mg diethylnitrosamine (DEN)/kg body wt. Thereafter chloroform was applied twice weekly for 11 consecutive weeks in doses of 100, 200 and 400 mg/kg body wt, respectively. This treatment raised the number of adenosine-5'-triphosphatase (ATPase)-deficient foci up to 5-fold, that of gamma-glutamyltranspeptidase (GGTase) and glycogen-positive foci 13- and 10-fold, respectively, after 12 weeks; 25 mg caused no effect compared to DEN-treated controls. In contrast, daily doses of chloroform only, 200 and 400 mg/kg body wt for 33 days, and 800 mg/kg body wt for 20 days given to 3-4-week-old female Sprague-Dawley rats did not lead to island formation, measured after 12 weeks, indicating a promoting rather than an initiating potency.

Dose-dependent promoting effect of polychlorinated biphenyls on enzyme-altered islands in livers of adult and weanling rats.

The effect of the technical mixture of polychlorinated biphenyls (PCBs) Clophen A 50 on the appearance of enzyme-altered islands initiated by diethylnitrosamine (DEN) in livers of 6 and 3 weeks old female Sprague-Dawley rats was studied. The loss of adenosine-5'-triphosphatase (ATPase), the emergence of gamma-glutamyltranspeptidase (GGTase), and the glycogen storage were used as histochemical markers. Islands were initiated by gastric intubation of 12 X 8 mg DEN/kg body weight/day in adults, or with 1 X 8 mg DEN/kg body weight in weanlings. Clophen A 50 alone initiated only few islands. A dose-dependent enhancement in number and area of islands by an additional treatment with Clophen A 50 of DEN-pretreated animals (2-100 mg/kg body weight/weekly, for 7 weeks) was observed in both age groups. In adults, doses between 2 and 100 mg/kg body weight increased number and area of ATPase-deficient islands 2 to 12-fold. In weanlings, application of 10-100 mg/kg body weight resulted in an increase of number and area up to 7- and 12-fold, respectively. No promoting effect was found with 2 mg/kg body weight compared to DEN-treated weanlings. The number of islands with coincidence of the three histochemical markers was enhanced dose-dependently in adults, and less marked also in weanlings after the application of the promoter.