Enhancement of N-nitrosodiethylamine-initiated hepatocarcinogenesis by phentoin in male F344/NCr rats at a dose causing maximal induction of CYP2B.

The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of male F344/NCr rats initiated with a single i.p. dose of N-nitrosodiethylamine (75 mg/kg b.w.) was studied. Beginning 2 weeks post-initiation, the rats received control diet or diet containing 500 or 1,500 ppm DPH or 500 ppm phenobarbital. At 52 weeks age, the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0%, 17% (1 +/- 0), 42% (1.8 +/- 0.8), or 67% (2.5 +/- 1.9) in rats exposed to N-nitrosodiethylamine alone, or the carcinogen followed by 500 ppm DPH, 1,500 ppm DPH, or 500 ppm phenobarbital, respectively. Between 53 and 79 weeks of age, 39% of rats receiving N-nitrosodiethylamine alone developed multiple (1.5 +/- 0.8) hepatocellular adenomas. A similar incidence (41%) occurred in the rats administered the carcinogen followed by 500 ppm DPH. The incidence of hepatocellular adenomas (88% and 89%) was significantly greater in rats exposed to N-nitrosodiethylamine followed by 1,500 ppm DPH or 500 ppm phenobarbital, respectively. Multiplicities of hepatocellular adenomas were significantly greater than the control value in rats fed 1,500 ppm DPH or 500 ppm phenobarbital (5.9 +/- 4.8 and 10.1 +/- 6.7, respectively), but not in the rats receiving 500 ppm DPH (2.3 +/- 1.6). No rats exposed to N-nitrosodiethylamine alone or the carcinogen followed by 500 ppm DPH developed hepatocellular carcinomas, while hepatocellular carcinomas occurred in 29% or 67% of the rats given 1,500 ppm DPH or 500 ppm phenobarbital, respectively, following initiation. Increases in hepatic CYP2B-mediated benzyloxyresorufin O-dealkylation activity in rats exposed to 500 and 1,500 ppm DPH for 2 or 23 weeks were approximately 50% and approximately 100%, respectively, of the maximal induction caused by 500 ppm phenobarbital. Thus, in the rat model, DPH enhanced N-nitrosodiethyl-amine-initiated hepatocarcinogenesis when administered at a dose causing maximal CYP2B induction.

Dose-response relationships for cytochrome P450 induction by phenobarbital in the cotton rat (Sigmodon hispidus).

The induction of a hepatic pleiotropic response, including increase in liver/body weight ratio, induction of hepatic CYP2B and CYP3A protein and catalytic activity, and hepatic microsomal epoxide hydration activity, was investigated in male cotton rats (Sigmodon hispidus) administered graded dietary concentrations (0-1500 ppm) of phenobarbital (PB) for 14 days. A dose-dependent induction of each endpoint was observed, although plateaus in the various dose-response curves were not obtained, and ED50 values (PB concentrations associated with half-maximal responses) for the various endpoints were not able to be calculated. A maximal 1.31-fold increase, compared to the control value, in live/body weight ratio was observed, while microsomal epoxide hydration activity was increased as much as 3.6-fold by PB administration. Pentoxy- and benzyloxyresorufin O-dealkylation and testosterone 16 beta-hydroxylation activities (considered to be relatively selective for CYP2B in the Norway rat (Rattus norvegicus)), were induced maximally less than five-fold. Testosterone 6 beta-hydroxylation (considered to be relatively selective for CYP3A in R. norvegicus) was induced maximally less than two-fold. Maximal induction of 7-ethoxy-4-trifluoromethyl-coumarin O-deethylation was 18-fold, compared to the control rate. Western blotting studies indicated that hepatic microsomal proteins immunoreactive with polyclonal antisera to R. norvegicus CYP2B1 or CYP3A1 were induced, in a dose-responsive manner, by PB in the cotton rats. These results indicate that the cotton rat responds to PB treatment with a coordinate pleiotropic response similar to that displayed by R. norvegicus, although the substrate specificity of the induced proteins appears to differ between the two rodent species.

Histogenesis and the role of p53 and K-ras mutations in hepatocarcinogenesis by glyceryl trinitrate (nitroglycerin) in male F344 rats.

Glyceryl trinitrate (GTN) was previously reported to induce hepatocellular carcinoma (HCC) in rats after prolonged feeding. The present experiments were undertaken to evaluate the histogenesis and molecular biology of these tumors and the possible role of nitric oxide (NO), a GTN metabolite, in their development. Male F344 rats received a single i.g. intubation of GTN (1.2 g/kg) at 6 weeks of age and/or a diet containing 1% GTN from 8 weeks of age until necropsy, i.e. for up to 78 weeks. Some animals were subjected to 2/3 partial hepatectomy (PH) at 9 weeks of age. Five sequential sacrifices (14, 32, 52, 78 and 84 weeks of age) were performed. No liver tumors developed in control rats or in rats that received GTN only by a single i.g. intubation, even when intubation was followed by PH. Preneoplastic foci, mainly of clear cell and mixed cell type (identified as positive for glutathione S-transferase placental form) were found from 14 weeks of age in rats receiving GTN in the diet. Focal eosinophilic areas (atypical foci) composed of atypical hepatocytes that often extended into the veins were observed beginning at 52 weeks of age. Some mixed hepatocholangiocellular adenomas and carcinomas arose in eosinophilic lesions. HCCs were seen beginning at 78 weeks of age, but only in rats receiving dietary GTN. Incidence of HCC in the latter animals was 50-75%. Most HCCs were well differentiated. The carcinogenic effect of GTN given in the diet was not affected by prior intubation of a large single dose followed by PH. No p53 mutations were found in 18 tumors but K-ras point mutations, all within codon 12, were found in 8/18 tumors, mostly those with cholangiocellular elements. These were first or second position G-->T transversions or second position G-->A transitions. While these mutation types have also been commonly seen in bacteria after NO-related DNA damage, the fact that tumors arose only on prolonged feeding of this potently bioactive agent at massive doses seems consistent with a more complex mechanism involving multiple (i.e. genetic and/or epigenetic) factors in carcinogenesis by GTN.

Enhancement of thyroid and hepatocarcinogenesis by 1,4-bis[2-(3,5- dichloropyridyloxy)]benzene in rats at doses that cause maximal induction of CYP2B.

To investigate the promoting effects of 1,4-bis[2-(3,5- dichloropyridyloxy)]benzene (TCPOBOP) on liver and thyroid carcinogenesis of rats at doses that cause maximal induction of hepatic CYP2B, 5-week-old male F344 rats were given either a single i.p. dose of 75 mg N-nitrosodiethylamine (NDEA)/kg body wt in saline or saline alone. After 2 weeks the rats were fed control diet or a diet containing 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. phenobarbital (PB; a positive control group). A total of four sequential sacrifices (9, 30, 52 and 79 weeks of age) was performed. At 30 weeks the mean volume (mm3) of hepatocellular foci in NDEA-initiated rats exposed to either dose of TCPOBOP or to PB was significantly increased as compared with rats exposed to NDEA followed by control diet (P < 0.05). In addition, the volume percentage of liver occupied by foci was significantly greater in NDEA-initiated/1000 p.p.m. TCPOBOP-promoted rats as compared with rats exposed to NDEA alone (P < 0.05, n = 6). At 52 weeks of age the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0, 83 (2.6 +/- 1.3), 100 (3.4 +/- 2.1) or 67% (2.5 +/- 1.9) in rats exposed to NDEA alone or NDEA followed by 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB respectively (n = 12). Hepatocellular carcinomas were found only in rats given 1000 p.p.m. TCPOBOP (17% incidence) or PB (8% incidence) following NDEA initiation. The incidences of thyroid follicular cell adenomas were 0, 17, 33 or 8% in rats exposed to NDEA alone or NDEA followed by 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB respectively. Between 53 and 79 weeks of age 38% of rats treated with NDEA alone developed multiple (1.5 +/- 0.8) hepatocellular adenomas. This incidence was enhanced to 100% in rats exposed to NDEA followed by either 330 or 1000 p.p.m. TCPOBOP. Multiplicities of hepatocellular adenomas were also increased significantly (10.5 +/- 3.9, 10.4 +/- 7.0 and 10.1 +/- 6.7 respectively) in rats promoted with 330 or 1000 p.p.m. TCPOBOP or 500 p.p.m. PB. None of the rats exposed to NDEA alone developed hepatocellular carcinomas, while multiple hepatocellular carcinomas occurred in 38% of the rats exposed to 330 p.p.m. and 78% of the rats given 1000 p.p.m. TCPOBOP following NDEA initiation. Thyroid follicular cell tumors occurred at 79 weeks in more than 40 and 50% incidences in rats exposed to NDEA followed by 330 or 1000 p.p.m. TCPOBOP respectively. Also, a significant decrease in serum levels of triiodothyronine and thyroxine were observed in non-initiated 79-week-old rats fed 1000 p.p.m. TCPOBOP, compared with age-matched untreated controls (n = 6). Increases in hepatic CYP2B-mediated benzyloxyresorufin O-dealkylase activity detected in rats exposed to 330 and 1000 p.p.m. TCPOBOP for 2 or 23 weeks were similar in magnitude to those caused by 500 p.p.m. PB. Thus TCPOBOP at maximal CYP2B induction doses exhibits a strong promoting activity for both liver and thyroid of rats.

Transplacental carcinogenesis by cisplatin in F344/NCr rats: promotion of kidney tumors by postnatal administration of sodium barbital.

Transplacental carcinogenic effects of cis-dichlorodiammineplatinum (cis-DDP) in F344 rats were investigated. Pregnant F344 rats were given a single i.p. administration of either cis-DDP (5 mg/kg body wt; group 1) or saline only (group 2) on Day 18 of gestation. Offspring of groups 1 and 2 were randomly and equally divided into two subgroups: 1a, 1b and 2a, 2b, respectively. Beginning at 4 weeks of age, offspring in groups 1b and 2b received 500 ppm of sodium barbital (NaBB) in diet, while those in groups 1a and 2a received normal diet. The experiment was terminated at 79 weeks of age. A low incidence (2/19; 10.5%) of male offspring exposed to transplacental cis-DDP (group 1a) developed renal cell adenomas. Postnatal administration of NaBB significantly enhanced this incidence (10/22; p < 0.01) in cis-DDP-initiated offspring. Also, multiple kidney tumors were more common in group 1b than any other group and three animals in this group developed frank renal cell carcinomas. cis-DDP, administered transplacentally, was a complete carcinogen for rat liver as the incidence of hepatocellular adenomas was significantly higher in offspring exposed transplacentally to cis-DDP than in those exposed to saline (20/82 vs 3/75; p < 0.05). NaBB, a known promoter of hepatocellular carcinogenesis in adult rats initiated with N-nitrosodiethylamine, failed to promote liver carcinogenesis initiated by transplacental cis-DDP. Tumors of the central nervous system (3/82; gliomas) and peripheral nervous system (2/82; schwannomas) were found only in offspring exposed transplacentally to cis-DDP. Thus, cis-DDP, administered transplacentally, was a strong initiator of renal cell tumors and a complete carcinogen for multiple organs in rat offspring.

Further evidence for promoter-dependent development of hepatoblastoma in the mouse.

In previous studies, we found that male D2B6F1 mice fed phenobarbital (PB) for 53 weeks following N-nitrosodiethylamine (NDEA) initiation developed a high (70-80%) incidence of malignant hepatoblastomas. A very low (3.3%) incidence of such tumors occurred in the absence of promoter treatment in NDEA-initiated mice observed for 60 weeks, although nearly 50% of these animals developed hepatocellular lesions. To investigate whether hepatocellular lesions in NDEA-initiated mice or spontaneous hepatocellular lesions promoted by PB in mice given PB but no NDEA, progress to hepatoblastomas later in life, mice exposed to NDEA alone and PB alone were maintained for 110 weeks. Hepatocellular tumors (adenomas and carcinomas) occurred in almost all (97%) mice given NDEA alone. However, only 10% of NDEA-treated mice developed hepatoblastomas. Thus, despite its ability to induce hepatocellular neoplasms, NDEA treatment alone was rarely sufficient to induce hepatoblastomas in these mice. In contrast, PB treatment in the absence of NDEA initiation promoted the development of spontaneously occurring hepatocellular lesions, a significant number (37%) of which progressed to hepatoblastomas. Our observations clearly show that in this animal model the development of hepatoblastoma from its precursor cells (hepatocellular adenoma and carcinoma cells) occurs predominantly in the presence of promoting agents such as PB.

Induction of cytochrome P-450 in Sigmodon hispidus (cotton rats) exposed to dietary Aroclor 1254.

The induction of immunoreactive cytochrome P-450 protein and associated catalytic activities in 10-wk-old male and female Sigmodon hispidus (cotton rats) exposed for 2 wk to low dietary levels of Aroclor 1254 (0.33, 1.0, 3.3, 10, and 33 ppm), or the prototype P-450 inducers phenobarbital, DDT, clotrimazole, and beta-naphthoflavone was examined. Ethoxy-(ETR), methoxy- (MTR), pentoxy- (PTR), and benzyloxyresorufin (BZR) O-dealkylation activities were significantly increased at 0.33 ppm Aroclor for males and 1.0 ppm for females, when compared to control levels. O-Dealkylation activities peaked at 3.3 ppm for males and 10 ppm for females. ETR and MTR O-dealkylation activities were increased four- to eightfold while PTR and BZR O-dealkylation activities increased only two- to threefold. Liver/body weight ratios also increased, with the maximum ratios observed at the highest Aroclor dose, and were associated with histopathologic hepatocyte lesions. While increases in liver/body weight ratio, immunoreactive CYP2B protein, and BZR O-dealkylation were detected following phenobarbital treatment, no increase in PTR O-dealkylation activity was observed. These results demonstrate that S. hispidus (both males and females) are extremely sensitive to low dietary levels of Aroclor 1254, responding with increases in liver/body weight ratio, immunoreactive P-450 protein, and O-dealkylation activities. The cotton rat would appear to be a sensitive feral target species for detecting exposure to certain environmental contaminants.

Promotion of hepatocellular foci and adenomas by di(2-ethylhexyl) phthalate and phenobarbital in C3H/HeNCr mice following exposure to N-nitrosodiethylamine at 15 days of age.

Previous studies have shown that phenobarbital (PB), as well as another known liver tumor promoter, alpha-hexachlorocyclohexane (HCH), inhibits hepatic tumor formation in infant N-nitrosodiethylamine (NDEA)-initiated C57BL/6 x C3H/He (B6C3F1) male mice. These inconsistencies in detecting PB and HCH as tumor promoters have raised important questions on the mechanism of tumor promotion in mice, as well as the reliability of the infant B6C3F1 mouse as an initiation model in two-stage carcinogenesis experiments. Therefore, in an effort to avoid the inconsistencies associated with the B6C3F1 mouse, the present study evaluated the ability of two known hepatic liver tumor promoters, di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, and phenobarbital (PB), a barbiturate, to promote hepatocellular tumorigenesis in mice of the C3H/HeNCr strain initiated during infancy. At 15 days of age, male and female C3H/HeNCr mice received either a single ip injection of NDEA (5 micrograms/g body weight) or saline. At weaning (4 weeks of age), mice were divided into 3 groups and treated with either DEHP in the diet (12,000 ppm), PB in the drinking water (500 ppm), or control drinking water and diet for 24 weeks. All mice were killed at 28 weeks of age and the number and size of hepatic foci and adenomas were evaluated. Mice exposed to NDEA+DEHP or NDEA+PB showed significant increases in the number and size of hepatic tumors compared to those receiving NDEA alone. DEHP treatment in males yielded larger adenomas than those seen in PB-treated males.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor promotion by an anticonvulsant agent, phenytoin, in mouse liver: correlation with CYP2B induction.

To investigate the liver tumor promoting effects of phenytoin (5,5-diphenylhydantoin; DPH), 5 week old male D2B6F1 mice were given a single i.p. dose of 90 mg N-nitrosodiethylamine (NDEA)/kg body wt in tricaprylin. Control groups received tricaprylin alone. After 2 weeks, the mice were given a diet containing 500, 250 or 125 p.p.m. DPH. Ten mice from each treatment group were killed at 30 weeks of age, at which time 10/10 mice given 500 p.p.m. DPH after NDEA initiation had developed multiple hepatocellular foci and adenomas. Such lesions were found only in 2/10 mice given NDEA alone. By 60 weeks, when the experiment was concluded, the incidences (and multiplicities, in units of tumors per tumor-bearing mouse) of hepatocellular adenomas were 60% (1.8 +/- 0.8), 100% (11.6 +/- 5.5), 77% (4.4 +/- 3.3) or 71% (2.6 +/- 1.3) in mice exposed to NDEA alone, or NDEA followed by 500, 250 or 125 p.p.m. DPH respectively. Hepatocellular carcinomas (87% incidence) and hepatoblastomas (33% incidence) were found only in mice given 500 p.p.m. DPH following NDEA initiation. Dose-dependent and profound increases in hepatic CYP2B-mediated benzyloxyresorufin O-dealkylase activity were detected in livers of B6C3F1 mice exposed for 14 days to dietary DPH (125, 250, 500 or 1000 p.p.m.). Similar increases in this activity were observed in D2B6F1 mice exposed to 500 and 250 p.p.m. for 30 or 60 weeks. Thus, increased hepatic CYP2B activity in mice exposed to DPH correlates with the tumor promoting effect of this compound.

Alterations in populations of GST-p-immunoreactive single hepatocytes and hepatocellular foci after a single injection of N-nitrosodiethylamine with or without phenobarbital promotion in male F344/NCr rats.

The fate of placental glutathione S-transferase (GST-P)-immunoreactive hepatocytes, detectable in livers of rats soon after treatment with N-nitrosodiethylamine (DEN), was examined sequentially with or without phenobarbital (PB) promotion. Group 1 male F344/NCr rats were administered a single i.p. injection of 200 mg DEN per kg body weight at 5 weeks of age. Group 2 rats were given 500 ppm PB in the diet two weeks after the DEN treatment. Groups of six rats were sequentially sacrificed 16, 42, 70, 126 and 238 days after DEN injection. In DEN-treated rats, GST-P immunoreactive hepatocytes (single cells and multiple cell foci) were detectable 16 days after DEN, the total numbers decreasing by day 70 and thereafter rising again. In the early stages the proportion of single immunoreactive hepatocytes was prominent, but with time a gradual increase in small GST-P+ hepatocellular foci and larger foci became evident. Feeding of PB to rats for 16-238 days after a single DEN injection resulted in increases of both single cells and foci, especially foci composed of more than three hepatocytes. The growth response was increasingly pronounced with time. Adenomas or carcinomas were only observed at 126 or 238 days. Numbers of GST-P+ foci far exceeded the numbers of foci visible in hematoxylin-eosin (H & E) stained sections, and a few H & E foci were negative for GST-P. Many GST-P+ foci smaller than ten cells were composed of histologically normal hepatocytes. Almost all GST-P+ foci identifiable in H&E stained sections were larger than ten cells, consisted of clear cells (in both groups) or mixed (clear-eosinophilic) cells in PB-exposed rats, and appeared to be evenly distributed throughout the three zones of the liver. These results suggest that the promotive effect of PB is most evident as an increase in larger hepatocyte populations composed of more than three GST-P+ hepatocytes, rather than in increasing the populations of single GST-P immunoreactive cells. PB may cause clonal expansion of these single GST-P reactive hepatocytes. This study provides evidence for the hypothesis that some of the GST-P reactive hepatocytes are initiated cells.

Progressive atypia in spontaneous and N-nitrosodiethylamine-induced hepatocellular adenomas of C3H/HeNCr mice.

The progression of hepatocellular adenomas to carcinomas has been less well documented in mice than in rats. We studied progression of spontaneous and chemically induced hepatocellular adenomas in male C3H/HeNCr mice by image analysis. Spontaneous lesions in 15, 18 and 21 month old untreated male C3H/HeNCr mice and experimentally induced lesions were examined. Experimental group 1 received a single i.p. injection of N-nitrosodiethylamine (DEN) (5 mg/kg body wt) at 15 days of age. Groups 2 and 3 were injected a second time with DEN at 15 or 20 weeks of age (75 mg/kg body wt), with interim sacrifices at 11, 16 and 34 weeks after the second DEN injection. Atypia in adenomas were classified into four grades according to cell size, tinctorial changes, cellular pleomorphism and trabecular pattern. At earlier stages of the neoplastic process (11 or 16 weeks after the second DEN dose), most adenomas were well-differentiated lesions with no atypia or focal grade 1 or 2 atypia. At later stages (34 weeks after the second DEN dose), a large proportion of hepatocellular tumors were classified as adenoma with grade 3 atypia or carcinoma. The proportion of carcinomas in mice treated with a second dose of DEN at 20 weeks of age was significantly higher than in mice treated with a single dose of DEN or in mice given a second dose of DEN at 15 weeks. A positive correlation was found between increase in the size of lesions and increased atypia in both spontaneous and DEN-induced lesions and with age for spontaneous tumors. These results support the hypothesis that mouse hepatocellular adenomas are truly neoplastic lesions in different stages of progression toward malignancy.

Effects of the oxazolidinedione anticonvulsants trimethadione and dimethadione and the barbiturate homolog 5,5-dimethylbarbituric acid on N-nitrosodiethylamine-initiated renal and hepatic carcinogenesis in the F344/NCr rat.

The oxazolidinedione anticonvulsant trimethadione (3,5,5-trimethyl-2,4-oxazolidinedione, TMO) as well as its major metabolite, dimethadione (5,5-dimethyl-2,4-oxazolidinedione, DMO), and a structural analog from the barbiturate series, 5,5-dimethylbarbituric acid (DMB), were fed to F344/NCr male rats previously given a single initiating injection of N-nitrosodiethylamine (NDEA). The known promoter, phenobarbital (5-ethyl-5-phenylbarbituric acid, PB), was employed in this study as a positive control. At dosage levels equimolar to 500 ppm PB, none of the three compounds promoted development of hepatocellular adenomas or carcinomas, in contrast to PB. The two oxazolidinedione analogs and DMB caused minimal or no induction of cytochrome P450 isozyme 2B1 (CYP2B1)-mediated alkoxyresorufin O-dealkylase activities following short-term (2 weeks) feeding to separate groups of 6-week-old male F344/NCr rats, in contrast to the dramatic induction caused by PB. Promotion of neither thyroid nor renal neoplasia was observed following prolonged feeding of any of the tested compounds, although a significantly higher frequency of premalignant renal cortical tubular lesions (dysplasias) was seen in rats exposed to TMO following NDEA initiation than in those treated with NDEA alone. These studies provide important additional data on structure/liver tumor promoting activity relationships, and yield further evidence that within this group of structurally related anticonvulsants, it is possible to separate anticonvulsant activity from tumor promoting activity in the rat liver.

Origin of spontaneous and transplacentally induced mouse lung tumors from alveolar type II cells.

Mouse lung tumors were induced transplacentally in offspring by treating C3H/HeNCrMTV- and Swiss Webster [Tac:(SW)fBR] mice during different periods of gestation with a single i.p. injection of N-nitrosoethylurea (ENU) at 0.5 mmol or 0.74 mmol/kg. Quantitative and qualitative evaluation of the lung tumors in the offspring at ages ranging from 1 week to 52 weeks was carried out by light microscopic study of hematoxylin and eosin-stained (H&E) serial and step sections. By nitroblue tetrazolium enzyme histochemistry, 3-hydroxybutyrate dehydrogenase (seen predominantly in Clara cells) was localized in frozen tissue sections. By avidin-biotin peroxidase complex immunohistochemistry, various specific cellular and nuclear markers were investigated on paraffin sections (antisera against surfactant apoprotein, Clara cell antigen, lysozyme, and 5-bromo-2' deoxyuridine). Normal lung and lung tumors were also studied by electron microscopy. A histological method was developed to assess all lesions present in the entire lung. It was shown that solid and papillary tumor types arose individually and that mixed solid/papillary forms represented a progression of the benign solid adenoma to the malignant papillary carcinoma. Immunocytochemical localization of DNA synthesis with 5-bromo-2' deoxyuridine gave the highest labeling indices at early stages of tumor growth. As the size of the papillary tumors increased, fewer nuclei were labeled/mm2 of tumor section. Lack of both specific Clara cell antigen and 3-hydroxybutyrate dehydrogenase and the absence of typical nonosmiophilic Clara cell granules indicated a cell of origin other than Clara cells. Evidence for alveolar type II cell origin of both solid and papillary neoplasms in spontaneous and induced tumors was found in the expression of surfactant apoprotein, the presence of mature lamellar bodies (solid tumors) or small lamellar bodies, and immature stages of lamellar bodies (papillary tumors). Lysozyme was present in mature alveolar type II cells and solid tumors but absent in fetal lung and papillary neoplasms. Tumors induced on gestation day 14 or day 16 had all developed by 2 weeks of age and generally did not increase in multiplicity with age, whereas those induced on day 18 showed a protracted development with regard to frequency, growth (size), and progression. The multiplicity of mouse lung tumors induced at different stages of fetal development paralleled the number of alveolar type II precursor cells (i.e., followed a bell-shaped pattern peaking on day 16 of gestation).

Dose response of hepatic and renal DNA synthetic rates to continuous exposure of bromodeoxyuridine (BrdU) via slow-release pellets or osmotic minipumps in male B6C3F1 mice.

We studied the use of acute and chronic 5-bromo-2'-deoxyuridine (BrdU) administration for detection of DNA-synthesizing cells in the liver and kidney of B6C3F1 male mice. Six-week-old mice were exposed to BrdU either acutely with a single-pulse (IP) injection 1 hr before sacrifice or chronically with the use of slow-release pellets or osmotic minipumps at one of four BrdU dose rates. Pellets (2.5, 10, 25, and 50 mg) and minipumps (2.5 and 10 mg equivalents) were implanted subcutaneously on the backs of the animals 4 or 7 days before sacrifice). BrdU incorporation into DNA was determined by immunohistochemistry using an anti-BrdU antibody. Mice chronically exposed to BrdU demonstrated increased levels of nuclear labeling compared with those receiving a single-pulse injection. No time-related increases in nuclear labeling were detected in hepatocytes or renal tubule cells of mice exposed to BrdU pellets and in the kidneys of mice receiving BrdU minipumps at the 7-day compared with the 4-day time point. In some cases, the labeling indices at 7 days were significantly decreased compared with those at 4 days. In contrast, a time-related increase in nuclear labeling was seen in hepatocytes and Kupffer cells of mice exposed to BrdU minipumps. Therefore, the method used to administer BrdU chronically to the animal appears to play an important role in presenting the true proliferative scenario in cell kinetic studies. Our findings also provide evidence for an effect of BrdU on normal proliferation rates in these tissues.

Carcinogenicity study of fecapentaene-12 diacetate on skin painting in SENCAR mice.

To investigate the effects of both diol esterification and coadministration with antioxidant on the tumorigenicity of fecapentaene-12 (FP-12) preparations, diacetylfecapentaene-12 (DAFP-12) in dimethylsulfoxide (DMSO) was applied to SENCAR mouse skin with or without the stabilizer, vitamin E, twice/week for 5 weeks, following which all animals were promoted for up to 25 weeks by weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). While positive controls receiving 7,12-dimethylbenz[a]anthracene (DMBA) instead of DAFP-12 in a similar protocol all developed papillomas (average of 23/animal), papilloma incidence in mice given DAFP-12 did not differ significantly from that of the vehicle control. We conclude that DAFP-12 shows little or no tumor initiating activity for mouse skin even when coadministered with vitamin E.

Persistence of 5-bromo-2'-deoxyuridine in tissues of rats after exposure in early life.

Outbred L10 rats received 4 subcutaneous injections of 3.2 mg per rat 5-bromo-2'-deoxyuridine (BrdUrd) at 1, 3, 7 and 21 days of age. Groups of rats were sacrificed 1 h, 1 month or 49 weeks after the final injection. Tissues were fixed in 70% ethanol, embedded in paraffin and unstained sections were prepared for immunohistochemical demonstration of BrdUrd. A monoclonal antibody to BrdUrd was used with the avidin biotin peroxidase-complex (ABC) technique. The numbers of immunoreactive nuclei were tissue, cell and time-related. The labeling indices declined from 1 day to 49 weeks for all tissues studied. At 49 weeks after the last exposure to BrdUrd, many cells were still reactive, especially in tissues with normal low cell turnover (brain, uterine stroma). For cells with high turnover, including lymphocytes and ovarian germinative epithelium, few or no labeled cells remained at 49 weeks. This study provides clear evidence for the persistence of BrdUrd in normal tissues, some of which may be targets for the carcinogenic effect of the chemical and others which are not targets.

Naturally-occurring age-dependent glutathione S-transferase pi immunoreactive hepatocytes in aging female F344 rat liver as potential promotable targets for non-genotoxic carcinogens.

Naturally occurring basophilic foci (focal hepatocellular proliferative lesions) (FHPL) in the livers of aging female F344/NCr rats could not be promoted to grow or progress into tumors after phenobarbital (PB) exposure. Instead, PB induced new unique eosinophilic hepatocellular foci and adenomas much quicker in old rats than in young rats. We now report that these foci are immunoreactive for glutathione S-transferase pi (GSTP) and that they appear to arise from some naturally occurring single and double GSTP-reactive cells and foci which occur spontaneously in the liver of aging F344 rats in an age-related fashion. PB and other nongenotoxic chemicals may act as "carcinogens" by promoting the growth (clonal expansion) of some of these putative spontaneously-initiated cells and foci into tumors.

Liver tumor promoters and other mouse liver carcinogens.

Mouse liver tumors are important end points for safety assessment of chemicals in rodent carcinogenicity assays. The mechanisms of induction of these tumors, whether by chemicals with significant genotoxic activities or by chemicals without such activities, remain unknown. If test mice have spontaneous tumors of high or low background incidence, the promotion of these tumors or of spontaneously initiated or susceptible cells may provide a basis for carcinogenesis induced by these compounds. Likewise, chronic toxicity and its associated target cell hyperplasia may also lead to promotion of these tumors. Experimental systems have been developed to study the mechanisms of tumor promotion in mouse liver. These models should significantly advance research into mechanisms of hepatocarcinogenesis and tumor promotion in mouse liver and may subsequently be used for human risk assessment.

Lack of promoting effect of clonazepam on the development of N-nitrosodiethylamine-initiated hepatocellular tumors in mice is correlated with its inability to inhibit cell-to-cell communication in mouse hepatocytes.

The tumor-promoting ability of clonazepam (CZP), a widely used benzodiazepine anticonvulsant, was investigated in an in vivo mouse liver tumor promotion assay and an in vitro mouse hepatocyte intercellular communication assay. The development of preneoplastic hepatocellular foci of cellular alteration and hepatocellular neoplasms was studied in male B6C3F1 mice initiated, at 5 weeks of age, with a single i.p. injection of N-nitrosodiethylamine (NDEA; 90 mg/kg body weight) in tricaprylin, followed by administration of either phenobarbital (PB; 0.05%) or CZP (0.068% or 0.136%) in diet beginning 2 weeks after carcinogen injection and continuing to 60 weeks of age. Several mice from each group were killed after 9, 21, 33 or 53 weeks on test diet, and portions of liver and other organs were fixed in formalin and examined histologically. Unlike PB, CZP did not promote the development of preneoplastic hepatocellular foci or neoplasms (adenomas and carcinomas) in NDEA-initiated mice. Following limited (2 weeks) dietary exposure at 0.15%, CZP was a potent inducer of hepatic P450IIB1-mediated alkoxyresorufin O-dealkylase activities. In contrast, the degree of induction in hepatic tissue from mice fed 0.136% CZP for 53 weeks was markedly lower than that in mice fed 0.05% PB for 53 weeks. In the in vitro assay, diazepam, a strong tumor promoter in mouse liver, significantly inhibited mouse hepatocyte gap junctional intercellular communication, while CZP had no significant effect on this parameter. Thus, CZP, a drug structurally related to diazepam, is inactive as a liver tumor promoter in mice.