Gender differences in serotype 2 adeno-associated virus biodistribution after administration to rodent salivary glands.

Salivary glands (SGs) have proven useful targets for clinical applications of gene therapeutics. In this toxicology and biodistribution study, which conforms to U.S. Food and Drug Administration Good Laboratory Practice regulations, four doses (10(7)-10(10) particles) of a serotype 2 adeno-associated viral (AAV2) vector encoding human erythropoietin were directly administered to the right submandibular gland of male and female BALB/c mice (n = 21 per gender dose group). Control-treated (saline administered; n = 66) and vector-treated (n = 168) animals did not differ in clinical appearance, morbidity and mortality rates, food and water consumption, weight gain ratios, and final weight. Clinical hematology values also were unaffected by AAV2 administration except for parameters influenced by the expression of the recombinant protein (e.g., hematocrit). Mice were killed on days 3, 30, 55, and 92. No major vector-related toxicity was uncovered after complete pathology and histopathology review. However, a significant gender-related difference in vector biodistribution was revealed by quantitative polymerase chain reaction. In male mice vector (group receiving 10(10) particles/animal) effectively transduced, and was primarily confined within, the SGs (i.e., approximately 800 times more copies in SGs than in liver; day 3) and long lived. In contrast, in female mice, SG transduction was less efficient (260-fold less than in males; day 3) and short lived, and vector was disseminated widely via both the bloodstream (SG:liver copy ratio, approximately 1) and saliva (30-fold greater than in males). The observed vector biodistribution is likely due to differences in AAV2 receptor targets and structural differences affecting SG integrity. Sexual dimorphism is a factor of major significance that could potentially affect gene therapy clinical applications in SGs.

Toxicologic evaluation of flavor ingredients added to cigarette tobacco: skin painting bioassay of cigarette smoke condensate in SENCAR mice.

Four comparative two-stage SENCAR mouse skin painting bioassays were conducted with cigarette smoke condensate (CSC) preparations to evaluate the effect of common American cigarette flavoring ingredients on tumor promotion. Each independent study employed a unique flavoring combination applied to tobacco at exaggerated levels, and in total resulted in an evaluation of 150 ingredients. Groups of 30-50 female SENCAR mice each were initiated topically with 50 microg of 7,12-dimethylbenz(a)anthracene (DMBA), and promoted thrice weekly for 26 weeks with either 10 or 20 mg of CSC from test cigarettes containing ingredient mixtures. For comparison, separate groups of mice received concurrent treatment with CSC from reference cigarettes prepared without added ingredients. Negative and positive controls were treated with acetone or 12-0-tetradecanoyl-phorbol-13-acetate (TPA) as a promoter, respectively. CSC-only groups served as promotion controls. Tumors developed in > 80% of the TPA-treated mice by study week 11, with a < 3% background tumor formation in the acetone treated controls at termination. Tumor incidence in CSC-only promotion control groups was < 20%, with no apparent difference between reference and test CSC groups. Approximately 70% of the DMBA-initiated mice promoted with 20 mg CSC developed tumors. Tumors first appeared around week 9, with about five tumors/tumor bearing animal. Tumor incidence, latency and multiplicity were CSC dose related, with a lower tumor incidence (approximately 50%), longer latency (12 weeks), and reduced tumor burden (four tumors/tumor bearing animal) at the 10 mg CSC dose level. While tumor incidence, latency and multiplicity data occasionally differed between test and comparative reference CSC groups, all effects appeared to be within normal variation for the model system. Furthermore, none of the changes appeared to be substantial enough to conclude that the tumor promotion capacity of CSC obtained from cigarettes containing tobacco with ingredients was discernibly different from the CSC obtained from reference cigarettes containing tobacco processed without ingredients.

Toxicity of 3,3',4,4'-tetrachloroazoxybenzene in rats and mice.

The toxicity of 3,3',4,4'-tetrachloroazoxybenzene (TCAOB) was evaluated in 13-week gavage studies in male and female F344/N rats and B6C3F1 mice. In addition to histopathology, evaluations included clinical chemistry, hematology, thyroid hormone analyses, and effects on sperm morphology and estrous cycle length. Groups of 10 rats and 10 mice of each sex were exposed to TCAOB at dose levels of 0, 0.1, 1, 3, 10, or 30 mg/kg 5 days a week for 13 weeks. In the rat studies, the major effects included death in the 30 mg TCAOB/kg dose group; at lower exposure levels, a decrease in body weight gain, a decrease in thymus weight, an increase in liver weight, an increase in hematopoietic cell proliferation in the spleen and liver, a responsive anemia, a decrease in platelet counts, a chronic active inflammation of the vasculature in the lung, an increase in cardiomyopathy, hyperplasia of the forestomach, and a marked decrease in circulating thyroxine concentrations were observed. In male rats a decrease in sperm motility in the epididymides was observed. In addition, in female rats an increase in lung, spleen, kidney, and heart weights and nephropathy was observed. Furthermore, the estrous cycle length was increased. In the mouse studies, the major effects for males and females included a decrease in thymus weights, an increase in liver and kidney weights, centrilobular hypertrophy in the liver, hematopoietic cell proliferation, hyperplasia of the forestomach, and dilatation of hair follicles. The spectrum of effects in both rats and mice after exposure to TCAOB indicates that dioxin-like effects occur in addition to effects that have not been observed with dioxin-like compounds. No no-observed-adverse-effect level was reached in male or female rats or mice.

Plasmid DNA malaria vaccine: tissue distribution and safety studies in mice and rabbits.

To evaluate the safety of a plasmid DNA vaccine, tissue distribution studies in mice and safety studies in mice and rabbits were conducted with VCL-2510, a plasmid DNA encoding the gene for the malaria circumsporozoite protein from Plasmodium falciparum (PfCSP). After intramuscular administration, VCL-2510 plasmid DNA was detected initially in all of the highly vascularized tissues, but at later time points was found primarily in the muscle at the site of injection, where it persisted for up to 8 weeks. After intravenous administration, plasmid DNA initially distributed at a relatively low frequency to all the tissues examined except the gonads and brain. However, plasmid DNA rapidly cleared, and by 4 weeks postadministration could be detected only in the lung of one of six animals evaluated. In a safety study in mice, eight repeated intramuscular injections of VCL-2510 at plasmid DNA doses of 1, 10, and 100 microg had no adverse effects on clinical chemistry or hematology, and did not result in any organ pathology or systemic toxicity. In a safety study in rabbits, six repeated intramuscular injections of VCL-2510 at plasmid DNA doses of 0.15 and 0.45 mg had no discernible effects on clinical chemistry, hematology, or histopathology. No evidence of autoimmune-mediated pathology, anti-nuclear antibodies (ANA), or antibodies to dsDNA were observed in the mouse or rabbit studies.

Toxicity of 3,3',4,4'-tetrachloroazobenzene in rats and mice.

The toxicity of 3,3',4,4'-tetrachloroazobenzene (TCAB) was evaluated in 13-week gavage studies in male and female F344/N rats and B6C3F1 mice. In addition to histopathology, evaluations included clinical chemistry, hematology, thyroid hormone analyses, and reproductive parameters. Groups of 10 rats and 10 mice of each sex were exposed to TCAB at dose levels of 0, 0.1, 1, 3, 10, or 30 mg/kg for 5 days a week for 13 weeks. In the rat studies, the major effects for both males and females included a 10% decrease in terminal body weight at 30 mg/kg/day, an increase in hematopoietic cell proliferation in the spleen at 10 and 30 mg/kg/day, and a responsive anemia at 10 and 30 mg/kg/day. A 15 to 30% decrease in platelet counts and a 20 to 40% decrease in thymus weights was observed at 10 and 30 mg/kg/day. An increase in liver weight up to 15% was found at 3 mg/kg/day and higher doses in males and at 10 and 30 mg/kg/day in females, respectively. An increase in spleen weights up to 15% was observed at 10 and 30 mg/kg/day in males and at 30 mg/kg/day in females. A marked decrease in circulating total thyroxine (TT4) was found in both males and females at all dose levels tested. TT4 could hardly be detected at 10 and 30 mg TCAB/kg/day. In addition, hyperplasia of the forestomach was increased at 3 mg/kg/day and higher doses in males and at 30 mg/kg/day in females. In the mouse studies, an increase in liver and spleen weight was observed up to approximately 25% in both males and females at 10 and 30 mg/kg/day. Hyperplasia of the forestomach was observed at 1 mg/kg/day and higher doses in both males and females. In males, a 30% decrease in thymus weights at 30 mg/kg/day and a 60% decrease in epididymal sperm density at 3 and 30 mg/kg/day was observed. Also in males, centrilobular hypertrophy of hepatocytes and an increase in hematopoietic cell proliferation in the spleen was observed at 3 mg/kg/day and higher doses. Based on the current study and information in the literature, TCAB has dioxin-like properties. Comparison of the effects of TCAB in the present study and in the literature to those with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) indicates that TCAB is from two to six orders of magnitude less potent than TCDD depending on the end point.

Glycine modulates the toxicity of benzyl acetate in F344 rats.

The influence of supplemental glycine on benzyl acetate (BA; a compound metabolized via the hippurate pathway)-induced toxicity was investigated. Groups of male F344 rats were fed NIH-07 diet containing 0, 20,000, 35,000, or 50,000 ppm BA for up to 28 days. Two additional groups were fed NIH-07 diet with 50,000 ppm BA and 27,000 ppm glycine or 50,000 ppm BA 32,000 ppm L-alanine; supplemental glycine and L-alanine were equimolar. The L-alanine group served as an amino nitrogen control. A third group was fed NIH-07 diet with 32,000 ppm L-alanine and served as an untreated isonitrogenous control BA caused increase in mortality, body weight loss, the incidence of abnormal neurobehavioral signs such as ataxia and convulsions, along with astrocyte hypertrophy and neuronal necrosis in the cerebellum, hippocampus, and pyriform cortex of the brain. These effects were reduced significantly by supplementation with glycine but not with L-alanine. These results suggest that the neurodegeneration induced by BA is mediated by a depletion of the glycine pool and the subsequent excitotoxicity.

The hepatocarcinogen methapyrilene but not the analog pyrilamine induces sustained hepatocellular replication and protein alterations in F344 rats in a 13-week feed study.

Methapyrilene (MPH) was a widely used antihistamine until it was found to produce hepatocellular carcinoma and cholangiocarcinoma in Fischer 344 rats. The structurally similar antihistamine pyrilamine (PYR) was marginally or noncarcinogenic in a similar study. The peroxisome proliferator Wy-14,643 was included in this study as a positive control. As part of a program to investigate the mechanisms whereby structurally similar chemicals produce different toxicities, we studied these three chemicals for the induction of cell proliferation in the liver of F344 rats. Male rats were treated for up to 13 weeks with feed dosed with MPH (HCl salt) at 0, 50, 100, 250, or 1000 ppm or PYR (maleate salt) at 1000 ppm to duplicate the route of administration and high-dose groups used in the carcinogenesis assay. In addition, the nongenotoxic hepatocarcinogen peroxisome proliferator Wy-14,643 was included as a positive cell-proliferating chemical. Cell proliferation was quantitated by measuring the incorporation of bromodeoxyuridine (BrDU) administered by osmotic minipump for 7 days and the appearance of proliferating cell nuclear antigen (PCNA) immunohistochemically. The BrDU-labeling index showed a large and sustained increase in rats treated with MPH at 250 and 1000 ppm, sustaining greater than 50% labeling in the higher dose group of 4-, 6-, and 13-week treatment groups. PYR at 1000 ppm demonstrated no significant increase in labeling above control levels at any time point. PCNA-labeling indexes showed similar but reduced increases for MPH and were comparable to control for the PYR dose groups. Two-dimensional gel electrophoresis was used for the detection of quantitative changes in gene expression and qualitative changes in the charges of specific mitochondrial and cytosolic proteins. Quantitative changes in 32 proteins induced by MPH and 39 changes induced by Wy-14,643 were detected throughout the 13-week study. Specific mitochondrial protein charge shifts were associated with high-dose MPH treatment that were not observed in animals treated with Wy-14,643. PYR induced no significant qualitative or quantitative protein alterations. Hepatocellular proliferation of the large magnitude observed following dietary administration of MPH, and not PYR may contribute to the mechanism of carcinogenesis of MPH.

Proliferation of carcinogen-damaged hepatocytes during cell-cycle-dependent initiation of hepatocarcinogenesis in the rat.

Hepatocyte proliferation and damage to DNA were characterized during the initiation phase of carcinogenesis in livers of rats that had received a single administration of the methylating agent methyl(acetoxymethyl)nitrosamine (DMN-OAc). Quiescent non-proliferating hepatocytes in intact livers did not appear to be susceptible to initiation by DMN-OAc, whereas proliferating hepatocytes in the S phase appeared to have greatest risk. To characterize the phenomenology of S-phase-dependent initiation further, the fractions of hepatocytes in the S and M phases of the cell cycle were enumerated at various times after treatment with DMN-OAc. Hepatocytes treated when in G1 experienced a delay of up to 20 h in the onset of S phase and a reduced rate of entry into the S and M cycle phases. Hepatocytes treated when in S phase experienced considerable delay in progression to mitosis due to part to inhibition of DNA replication. Hepatocytes treated when in late S/G2 also demonstrated a delay in progression into mitosis. The levels of 7-methylguanine and O6-methyldeoxyguanosine were quantified in the nuclear DNA of proliferating hepatocytes. The kinetics of removal of these lesions appeared to be first-order (half-life = 24 h). Hepatocyte risk of initiation was modeled by a function which summed over time the product of the fraction of hepatocytes in the S phase and the fraction of residual, unrepaired damage to DNA. For hepatocytes treated when in early G1, the time-weighted frequency of premutagenic DNA damage that was present during DNA replication was estimated to be less than half of that for hepatocytes treated when in early S. The results suggest that cell-cycle-dependent variation in sensitivity to initiation of hepatocarcinogenesis may be, in part, due to efficient removal of potentially carcinogenic lesions from DNA during an extended G1. The apparent high sensitivity of hepatocytes in late S/G2 suggests the contribution of additional factors.

Inhibition of initiator-promoter-induced skin tumorigenesis in female SENCAR mice fed a vitamin A-deficient diet and reappearance of tumors in mice fed a diet adequate in retinoid or beta-carotene.

Retinoids have chemopreventive activity for epithelial tumors in a variety of systems, including the two-stage tumorigenesis system of mouse skin in which only the promotion stage is inhibited. We asked whether dietary vitamin A deficiency could affect the skin tumorigenic response, prior to major changes in body weight or general health of the animals. Two regimens were tested to induce vitamin A deficiency. SENCAR mice were either (a) fed a vitamin A-deficient diet from 4 or 9 weeks of age or (b) their mothers were fed the diet from the time of birth of the experimental animals which were then weaned on the same diet. The latter regimen produced typical symptoms of vitamin A deficiency in the offspring by Weeks 12-14 and all the mice died by Week 19; the former regimen permitted sufficient accumulation of retinol and its esters to sustain life for up to 45 and 75 weeks, respectively, in the majority of mice. For our experiments, vitamin A depletion was produced by placing the mothers on the deficient diet at birth of the experimental animals. A single topical dose of 20 micrograms of 7,12-dimethylbenz(a)anthracene (DMBA) was used as the initiator at 3 weeks of age and 1 to 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) once weekly as the tumor promoter for 10 weeks (from Week 4 through 13 of the experiment). Fifty-five % of mice (n = 40) on Purina laboratory chow (mean body weight, 31.4 g) developed skin tumors (2.58 per mouse) at 12 weeks, versus 2.5% (0.05 papillomas per mouse) of mice (n = 40) kept on the purified vitamin A-deficient diet (mean body weight, 30.3 g), a 98% decrease in tumor/mouse. Retinoic acid (RA) (1-3 micrograms/g diet) supplementation after Week 12 caused a rapid tumorigenic response in 95% of the mice by week 22. This tumor response occurred to a reduced extent in the absence of continued TPA treatment up to Week 13. Even though tumor incidence increased within 1 week of RA and 95% of the mice showed the tumorigenic response, the number of tumors per mouse was about 50% of that observed in mice maintained on standard Purina diet. This was confirmed in an experiment in which the mice were maintained for life either on Purina or on the RA (3 micrograms/g) containing purified diet, the latter being the control group for the effect of vitamin A deficiency on skin tumorigenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Cadmium carcinogenesis in male Wistar [Crl:(WI)BR] rats: dose-response analysis of effects of zinc on tumor induction in the prostate, in the testes, and at the injection site.

The ability of zinc acetate to modify the carcinogenic effects of CdCl2 in male Wistar [Crl:(WI)BR] rats was studied over a 2-year period. Groups of rats received a single s.c. injection of Cd (30.0 mumol/kg) in the dorsal thoracic midline or i.m. in the right thigh at time 0. Zinc was given in three separate s.c. doses of 0.1, 0.3, or 1.0 mmol/kg (at -6, 0, and +18 h relative to cadmium) in the lumbosacral area or p.o. at 100 ppm in the drinking water (-2 to +100 weeks). Cadmium treatments (s.c.) resulted in the appearance of tumors at the injection site and in the testes. The incidence of s.c. injection site tumors (mostly mixed sarcomas) was markedly reduced by high dose (1.0 mmol/kg) s.c. zinc (50% reduction) and was almost abolished by p.o. zinc (92% reduction). Testicular tumors (mostly Leydig cell adenomas) induced by s.c. cadmium were reduced in a dose-related fashion by zinc and were found to be highly dependent on the ability of zinc to prevent the chronic degenerative effects of cadmium in the testes. Oral zinc had no effect on s.c. cadmium-induced testicular tumors, while i.m. cadmium alone did not induce these tumors. In rats in which s.c. cadmium-induced testicular tumors and chronic degenerative effects were prevented by zinc (1.0 mmol/kg, s.c.), a marked elevation in prostatic tumors (exclusively adenomas) occurred (control, 9.6%; cadmium plus high zinc 29.6%). Cadmium given i.m., which did not result in testicular tumors or degeneration, also induced an elevated incidence (42.3%) of prostatic tumors, again indicating a dependence on testicular function. Prostatic tumor incidence was also significantly elevated (25.0%) in rats receiving 1.0 mmol/kg zinc, s.c., in combination with i.m. cadmium. These results indicate that zinc inhibition of cadmium carcinogenesis is a complex phenomenon, depending not only on dose and route but also on the target site in question.

Infrequent activation of K-ras, H-ras, and other oncogenes in hepatocellular neoplasms initiated by methyl(acetoxymethyl)nitrosamine, a methylating agent, and promoted by phenobarbital in F344 rats.

Fischer 344/Ncr rats of both sexes were subjected to partial hepatectomy and then initiated 21-24 h later by a single injection of methyl(acetoxymethyl)nitrosamine at 0.1 mmol/kg body weight via the portal vein. Beginning 3 weeks later, development of hepatocellular neoplasms in initiated rats was promoted by feeding 0.05% phenobarbital (PB) in the diet. Not only intrahepatic lesions but also a variety of extrahepatic tumors were induced. High-molecular-weight DNAs were prepared from 67 samples of grossly normal liver containing multiple preneoplastic foci/areas of microscopic dimensions, 137 hepatocellular adenomas (nodules), 93 hepatocellular carcinomas (HCC), 10 cholangiomas, and 25 extrahepatic tumors in 95 rats and tested for transforming activity in the NIH 3T3 transfection assay. DNA preparations from 7 of 93 HCCs, 2 of 10 cholangiomas, 2 of 137 nodules, 1 histiocytic sarcoma, and 1 thyroid carcinoma were positive in the transfection assay. Southern blot analysis showed that NIH 3T3 transformants induced by DNA from 5 HCCs, 1 hepatocellular adenoma, 1 cholangioma, 1 histiocytic sarcoma, and 1 thyroid carcinoma contained an activated K-ras gene of rat origin. Rat-derived H-ras was identified in transformants from 2 additional HCCs and rat c-raf from 1 hepatocellular adenoma. The transforming gene from one cholangioma showed no sequence homology to the ras genes, neu, or c-raf. Immunoprecipitation analysis of ras Mr 21,000 protein in 11 transformants indicated that, based upon protein electrophoretic mobilities, activation of the ras genes consistently resulted from mutations in codon 12 of these genes. Selective oligonucleotide analysis revealed that a G----A transition in the second base of codon 12 of K-ras was present in the 9 K-ras-positive transformants and also in DNAs prepared from the original tumors. In contrast, oligonucleotide hybridization experiments with DNAs from 35 hepatocellular tumors that were negative in transfection assays revealed the presence of mutant K-ras in 1 of 15 HCCs; no mutation could be detected in 20 transfection-negative adenomas. The infrequency of detection of a specific oncogene, more frequent detection of oncogenes in malignant tumors, and failure to observe activated oncogenes in preneoplastic lesions suggest that activation of ras oncogenes may occur as a late and infrequent event in the evolution of some rat hepatocellular neoplasms and that mutation of a specific ras locus is not an obligatory early event in the genesis of these neoplasms.

Cadmium carcinogenesis in male Wistar [Crl:(WI)BR] rats: dose-response analysis of tumor induction in the prostate and testes and at the injection site.

Carcinogenic dose-response effects of CdCl2 in male Wistar [Crl:(WI)BR] rats were studied over a 2-year period. Groups of rats received a single s.c. injection of CdCl2 at doses of 0, 1.0, 2.5, 5.0, 10.0, 20.0, or 40.0 mumol/kg in the dorsal thoracic midline. Other groups received either four separate s.c. doses of 5 mumol Cd/kg each (at 0, 48, 96, and 168 h), or low dose cadmium (5.0 mumol/kg, s.c., at 0 h) followed by a higher dose (10.0 or 20.0 mumol/kg, s.c., at 48 h). The cadmium treatments resulted in appearance of tumors at the injection site, in the testes, and in the ventral prostate. Injection site tumors (mostly sarcomas) appeared to be strictly related to accumulated dose of cadmium and approached a 45% incidence at the highest cadmium dose (40 mumol/kg). Testicular tumors (mostly Leydig cell adenomas) were found to be highly dependent on testicular degeneration caused by cadmium. The highest Leydig cell tumor incidence occurred in the 40 mumol/kg (83%) and 20 mumol/kg (72%) dosage groups. Low dose pretreatment (5.0 mumol/kg) reduced or prevented the testicular degeneration and tumor formation that would otherwise result from a subsequent higher dose of CdCl2 (20 mumol/kg). Prostatic tumors (mostly adenomas of the ventral lobe) were also found to be associated with cadmium treatment, but in a non-dose related fashion. Prostatic tumor incidence was significantly elevated at the 2.5 mumol/kg dose of CdCl2 (eight tumors/26 rats; 31%) and showed a strong positive correlation between 0.0 and 2.5 mumol/kg in both tumor incidence and multiplicity. At higher doses, including those that caused marked testicular degeneration and induced prostatic atrophy, an elevated incidence of tumors did not occur. The occurrence of hyperplastic foci of the prostate, however, showed a strong positive correlation with increasing dose after single injections of cadmium up to and including 20.0 mumol/kg. Results indicate that CdCl2 can induce preneoplastic lesions of the prostate that appear to develop into tumors only at doses well below those causing marked degeneration of the testes and atrophy of the prostate.

Activated neu oncogene sequences in primary tumors of the peripheral nervous system induced in rats by transplacental exposure to ethylnitrosourea.

Neurogenic tumors were selectively induced in high incidence in F344 rats by a single transplacental exposure to the direct-acting alkylating agent N-ethyl-N-nitrosourea (EtNU). We prepared DNA for transfection of NIH 3T3 cells from primary glial tumors of the brain and from schwannomas of the cranial and spinal nerves that developed in the transplacentally exposed offspring between 20 and 40 weeks after birth. DNA preparations from 6 of 13 schwannomas, but not from normal liver, kidney, or intestine of tumor-bearing rats, transformed NIH 3T3 cells. NIH 3T3 clones transformed by schwannoma DNA contained rat repetitive DNA sequences, and all isolates contained rat neu oncogene sequences. One schwannoma yielded a transformant with rat-specific sequences for both neu and N-ras. A point mutation in the transmembrane region of the putative protein product of neu was identified in all six transformants and in the primary tumors from which they were derived as well as in 5 of 6 schwannomas tested that did not transform NIH 3T3 cells. Of 59 gliomas, only one yielded transforming DNA, and an activated N-ras oncogene was identified. The normal cellular neu sequence for the transmembrane region, but not the mutated sequence, was identified in DNA from all 11 gliomas surveyed by oligonucleotide hybridization. Activation of the neu oncogene, originally identified [Schechter, A.L., Stern, D.F., Vaidyanathan, L., Decker, S.J., Drebin, J.A., Greene, M.I. & Weinberg, R.A. (1984) Nature (London) 312, 513-516] in cultured cell lines derived from EtNU-induced neurogenic tumors that by biochemical but not histologic criteria were thought to originate in the central nervous system in BD-IX rats, appears specifically associated with tumors of the peripheral nervous system in the F344 inbred strain.

Anomalous growth of rat incisor teeth during chronic intermittent vitamin A deficiency.

Rapid, synchronous and repeating cycles of marginal retinoic acid sufficiency and deficiency were produced in rats by following an established protocol. During cycles 11-15 (343-464 days after weaning), 17 of 24 rats (71%) developed tumor-like masses in the connective tissue surrounding the formative ends of one or both upper incisor teeth. The masses were composed of cords of odontogenic epithelium surrounded by a mantle of mesenchyme. Lakes of predentine, associated with foci of keratinized epithelium, were randomly distributed. On the basis of current understanding of odontogenesis, we propose that periodically disturbed differentiation of pulpal mesenchymal cells to dentin-secreting odontoblasts was pivotal to development of the masses. During deficiency, dentin secretion is impaired, the dentin wall perforates and with time the odontogenic epithelium and pulp herniate into the surrounding connective tissues. We propose that the incisal masses arose because the pulpal mesenchyme continued to grow and its secretion product, dentine, continued to be deposited (during periods of vitamin A sufficiency) in an ectopic site where functional attrition from mastication could not occur.

Cell cycle-dependent initiation of hepatocarcinogenesis in rats by methyl(acetoxymethyl)nitrosamine.

Hepatocyte sensitivity to initiation of carcinogenesis was studied as a function of the cell cycle phase in which damage was incurred. Hepatocytes were stimulated to proliferate by a two-thirds partial hepatic resection, and their proliferation was synchronized further by postsurgical treatment with hydrocortisone. Groups of male F344 rats were given a single administration of methyl(acetoxymethyl)nitrosamine, a highly reactive methylating agent, at various times after two-thirds partial hepatic resection when hepatocytes were in defined phases of the cell cycle. Beginning 3 wk after the treatment and for 37 wk thereafter, rats were fed a diet containing 0.05% phenobarbital to promote the expression of initiated hepatocytes. At 45 wk after treatment with carcinogen hepatocytic neoplasms were enumerated. The greatest yield of neoplasms (5.4 per liver) was observed in the group treated 16 h after two-thirds partial hepatic resection or at the time when proliferating hepatocytes began to enter the S phase of the cell cycle. The least yield of neoplasms (0.8 per liver) was identified in the group treated with methyl(acetoxymethyl)nitrosamine when hepatocytes were early in G1. In the proliferating hepatocytes sensitivity rose continuously during G1 to a peak at the G1-S border and then fell continuously as hepatocytes traversed S, G2, and M. This pattern of response could not be attributed to variation in hepatic esterase which activates methyl(acetoxymethyl)nitrosamine or to variation in methylation of DNA. The results support a model in which carcinogen-induced genetic alterations, occurring at the time of or soon after damaged cells enter the S phase, represent irreversible events that contribute to the initiation of carcinogenesis.

Differential carcinogenic effects of intraperitoneal initiation with 7,12-dimethylbenz(a)anthracene or urethane and topical promotion with 12-O-tetradecanoylphorbol-13-acetate in skin and internal tissues of female SENCAR and BALB/c mice.

Groups of female SENCAR or BALB/c mice were initiated once intraperitoneally with 300 micrograms/mouse of 7,12-dimethylbenz(a)anthracene (DMBA) or 20 mg/mouse of urethane at 7 weeks of age. Beginning one week later, mice received topically applied acetone or 12-O-tetradecanoylphorbol-13-acetate (TPA), once weekly, at 2.5 micrograms/mouse for weeks 1 through 6 and 1.25 micrograms/mouse for weeks 7 through 52. The skin lesions were evaluated clinically. A complete necropsy was performed on all mice at week 52. SENCAR mice exposed to DMBA/TPA and urethane/TPA had more skin tumors than SENCAR mice exposed to DMBA or urethane alone and more than BALB/c mice in any treatment group. Of all skin carcinomas diagnosed histologically in DMBA/TPA-exposed mice, less than one-third had been identified clinically while the mice were alive. Most of the carcinomas arose within papillomas. BALB/c mice developed more vascular and uterine tumors than did SENCAR mice injected with DMBA and more lung and vascular tumors than did SENCAR mice injected with urethane. TPA exposure after treatment with either initiator had no significant effect on internal tumor development in either SENCAR or BALB/c mice.

Pathology of aging female SENCAR mice used as controls in skin two-stage carcinogenesis studies.

The pathology of 60 aged female SENCAR mice used as acetone controls in skin painting studies was studied. Fifty percent of the mice survived past 96 weeks of age. The major contributing causes of death identified in 42 mice were glomerulonephritis (8 mice), histiocytic sarcoma (7 mice), and other tumors (8 mice). Glomerulonephritis was found in the majority of mice and was associated with thymic hyperplasia, focal vasculitis, and lymphoid hyperplasia. Necropsy of 58 mice surviving past 50 weeks of age revealed that 41 had an average of 1.36 tumors per mouse. The most common tumors included histiocytic sarcoma (13 mice), pulmonary adenoma or adenocarcinoma (11 mice), mammary tumors (11 mice), follicular center cell lymphoma (4 mice), and hepatocellular adenoma (4 mice). The 13 histiocytic sarcomas appeared to arise in the uterus and metastasized to liver (9 mice), lung (4 mice), kidney (3 mice), and other tissues. Lung tumors were of the solid and papillary types, and tumor cells frequently contained surfactant apoprotein (SAP) but did not contain Clara cell antigens, suggesting their origin from alveolar Type II cells. A variety of nonneoplastic lesions, similar to those observed in other mouse strains, were seen in other tissues of these mice. Amyloid-like material was seen only in nasal turbinates and thyroid gland. In a group of 28 mice exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) for up to 88 weeks, as a control for other treatment groups, 7 (25%) had papillomas and 5 (17.8%) had squamous cell carcinomas of the skin at necropsy, although many other induced papillomas regressed during the study.

Activated K-ras and N-ras oncogenes in primary renal mesenchymal tumors induced in F344 rats by methyl(methoxymethyl)nitrosamine.

Administration of methyl(methoxymethyl)nitrosamine to newborn Fischer 344 rats results in the preferential induction of renal tumors arising from the mesenchymal component of the kidney. DNA from a significant proportion of these tumors was capable of transforming NIH/3T3 cells. This report describes the renal tumor model, the detection of two different ras transforming genes in the kidney tumors (the N-ras oncogene in 1 and K-ras oncogene in 10 kidney tumors) and the characterization of DNA sequences specifying the transformed phenotype.

Ethionine carcinogenesis in CD-1, BALB/c and C3H mice.

In two separate in vivo studies, ethionine was evaluated for carcinogenic activity in mice. In the first study, DL-ethionine was fed in a chow diet at 0 (controls), 0.1 (low dose, LD) and 0.25% (high dose, HD) concentrations to the following groups of mice (30 animals/group): Swiss Webster CD-1 females, BALB/c males, and C3H/HeN males and females. Because of severe toxicity, BALB/c females were fed 0.05% (LD) and 0.1% (HD) ethionine. The Swiss and BALB/c mice were maintained on their respective diets for up to 105 weeks before killing whereas the C3H mice were killed at 68 weeks because of the high spontaneous incidence of liver tumors in this strain. The percentages of animals at risk (surviving the time to the first liver tumor recorded in each sex and strain) that bore liver tumors were as follows: Swiss female control, 0% (0/29), Swiss female LD, 87% (20/23); Swiss female HD, 89% (16/18); C3H male controls, 35% (8/23); C3H male LD, 55% (16/29); C3H male HD, 58% (15/26); C3H female controls, 5% (1/20); C3H female LD, 60% (12/20); C3H female HD, 92% (12/13); BALB/c male controls, 4% (1/23); BALB/c male LD, 8% (2/24); BALB/c male HD, 31% (5/16); BALB/c female controls, 0% (0/30); BALB/c female LD, 52% (14/27); and BALB/c female HD, 92% (12/13). The female mice were more responsive than the males in developing liver tumors. The results of the feeding study are compared with those obtained in a second study in which C3H female mice were intubated with 0, 150 or 500 mg DL-ethionine/kg body wt three times per week for 30 weeks and killed at 2 years. Only the LD mice showed a significantly increased incidence of liver tumors (20/39) as compared to controls (12/41) or HD mice (7/37) in the latter study. The hepatic levels of the major ethionine metabolite and methylase inhibitor, S-adenosylethionine (AdoEt), as well as of the endogenous methyl group donor, S-adenosylmethionine (AdoMet) were determined in Swiss female mice fed either 0.1 or 0.3% in the diet for 1-6 weeks. Hepatic AdoEt levels ranged from 37 to 80 micrograms/g liver in the LD animals and from 61 to 203 micrograms/g liver in the HD group; levels of the endogenous metabolite AdoMet correspondingly dropped to 65% of the normal levels. The present results (i) extend to different strains and to both sexes previous observations demonstrating the hepatocarcinogenic activity of ethionine in mice; and (ii) indicate that as in the rat such activity may be exerted through the formation of AdoEt.

Effects of short-term exposure to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate on skin carcinogenesis in SENCAR mice.

Skin tumor promotion after a short-term exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied in female SENCAR mice. Mice were dosed once by the topical application of 20 micrograms of dimethylbenz[a]anthracene (DMBA) in 0.2 ml acetone. A week later, they received topical applications of TPA (2 or 4 micrograms per 0.2 ml acetone) once or twice a week for periods of 1-10 weeks and were killed at 30 weeks. Skin tumors were counted and measured for size weekly. When TPA was applied once a week for 10 weeks or only twice a week for 2 weeks, there was significant promotion of papilloma formation in a large proportion of mice initiated with DMBA. Mice that received one or two applications had a few skin tumors. The total number of papillomas decreased considerably and the majority appeared to regress after 20 weeks in mice that received TPA treatment for 10 weeks. In mice that received only 4 TPA treatments, however, the majority of the papillomas grew progressively in size and did not regress during the entire experimental period. A greater proportion of these tumors progressed to carcinoma than did those in mice receiving TPA for 10 weeks. Thus, a short-term exposure was effective in causing certain changes in skin of SENCAR mice that led to tumor development and progression.