13-cis-retinoic acid: inhibition of bladder carcinogenesis in the rat.

Transitional cell and squamous cell cancer of the bladder was induced in Wistar/Lewis female rats by direct instillation of N-methyl-N-nitrosourea into the bladder. Feeding of the synthetic retinoid, 13-cis-retinoid acid, inhibited the incidence and extent of bladder cancer in these rats, even when 13-cis-retinoic acid administration was begun after completion of the carcinogen treatment.

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.

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.

Preventive and enhancing effects of retinoids on the development of naturally occurring tumors of skin, prostate gland, and endocrine pancreas in aged male ACI/segHapBR rats.

The effects of dietary retinoids on the development of naturally occurring tumors in retired breeder male ACI/segHapBR rats were investigated. Groups of rats (21-25 mo of age, an age when early neoplasms first appear and tumor incidences are generally low) were fed diets containing 1 of 3 retinoids--all-trans-N-4-(4-hydroxyphenyl)retinamide (4-HPR), 783 mg/kg diet; all-trans-N-(4-pivaloyloxyphenyl)retinamide (4-PPR), 951 mg/kg; or all-trans-4-N-(2-hydroxyethyl)retinamide (2-HER), 687 mg/kg--or control diet for up to 54 weeks (average, 33 wk). Rats were maintained until less than 20% remained and the experiment was terminated. Contributing causes of death were determined, and a complete necropsy was performed for each rat. There was no difference between the retinoid-treated rats and control rats in the average age at death (30-31 mo) or in the average experimental survival time (29-35 wk), in the proportions of tumor-bearing rats (95.6-100%), or in the average number of organs with tumor per rat (2.1-2.5). The incidences of pancreatic islet cell adenoma and skin tumors were significantly different between control and some retinoid-treated groups. 4-PPR and 2-HER significantly enhanced pancreatic islet cell adenoma yields (P less than .025 and 0.05, respectively) whereas 4-HPR significantly inhibited epithelial and connective tissue skin tumor yields (P less than .025). Incidences of skin and prostate tumors were lower than in controls, but not significantly, in rats receiving 4-PPR and 2-HER. Most of the islet cell adenomas were shown, by avidin-biotin-peroxidase complex immunocytochemistry, to be insulinomas. 4-HPR would seem to be the most effective retinoid in the group, inasmuch as it prevented skin tumor development, may have slightly decreased the incidence of prostate tumors, and did not enhance islet cell tumor incidence.

Expression of RNA of an endogenous replication-defective retrovirus in rat mammary adenocarcinomas induced by 7,12-dimethylbenz[a]anthracene.

An investigation into the possible relationship between chemical carcinogen induction of rat mammary tumors and the expression of an endogenous retroviral genome was initiated. Mammary tumors were induced in female SD rats with 7,12-dimethylbenz[a]anthracene (DMBA). Tumors, identified histologically as mammary adenocarcinomas, were analyzed for RNA of a replication-defective endogenous retrovirus or RNA of a helper-independent endogenous type C virus. Expression of RNA of the replication-defective virus was detected in mammary tumors weighing 0.2--2.0 g. Larger tumors, for which histologic examination revealed proportionally more fibroblastic tissue than epithelial cells, did not contain comparable concentrations of this viral RNA. RNA homologous to a helper-independent rat type C retrovirus was not detected in tumors of any size. A cell line was established from a primary DMBA-induced mammary adenocarcinoma and appeared similar to the small mammary tumors with respect to endogenous type C viral RNA expression. We discuss possible implications of the expression of endogenous replication-defective viruses for use as markers for the effects of chemical carcinogens.

Dose response to intrarectal administration of N-methyl-N-nitrosourea and histopathologic evaluation of the effect of two retinoids on colon lesions induced in rats.

Male F344 rats, 8 weeks of age, were given 16 intrarectal administrations of N-methyl-N-nitrosourea (NMU) at one of three dose levels over a period of 8 weeks. Five days after the final NMU instillation, rats were placed on one of three diets: chow with gelatin beadlets, chow with beadlets containing 0.024% 13-cis-retinoic acid, or chow and beadlets with 0.006% of the trimethylmethoxy phenyl analog of retinoic acid ethylamide. Groups of 20-40 rats were killed at 22-26 weeks after the first carcinogen treatment. The number of rats with colon carcinoma and the number of tumors per rat were dose related. In addition, "blind" histopathologic evaluation of four predesignate colon locations revealed a dose-related incidence of microscopic preinvasive and invasive colon carcinomas. The feeding of diets containing these two retinoids did not significantly alter the incidence of these parameters of carcinogenesis or the mean histopathologic score at predesignated colon locations for preinvasive or invasive neoplastic lesions. Over 90% of the colon neoplasms induced were invasive tubulopapillary adenocarcinomas. The diameters of the tumors correlated significantly with degrees of invasion of the colons. Only 1 tumor (a signet ring carcinoma) metastasized to the peritoneal cavity. Only 2 of 300 rats treated with NMU had tumors at sites other than the colon.

Selective induction of intestinal tumors in rats by methyl(acetoxymethyl)nitrosamine, an ester of the presumed reactive metabolite of dimethylnitrosamine.

Methyl(acetoxymethyl)nitrosamine (DMN-OAc) was synthesized and tested for toxicity and carcinogenicity in rats to test the hypothesis that alpha-hydroxylation is required for metabolic activation of dimethylnitrosamine (DMN) to a reactive, proximate carcinogen. The acute median lethal doses (LD50) of DMN-OAc and DMN injected ip into 5-week-old male Sprague-Dawley (Charles River (CD) rats were determined to be 0.19 and 0.59 mmole/kg body weight or 25 mg DMN-OAc/kg and 44 mg DMN/kg body weight, respectively. Single ip injections of one-half the LD50 DMN-OAc (13 mg/kg body weight) in 5-week-old rats of both sexes resulted in a high incidence of epithelial tumors of the intestinal tract. Mean survival times for rats with intestinal tumors were 353 days for males and 433 days for females. Tumors were rarely found at other sites. DMN at equivalent toxic (one-half the LD50, 22 mg/kg) and molar (= one-sixth LD50, 7.0 mg/kg) dose levels, yielded (as expected) tumors of kidneys, lungs, and occasionally other organs, but at a much lower incidence. The finding of the potent carcinogenicity of DMN-OAc supported the postulate that alpha-hydroxylation of DMN in vivo generates a proximate carcinogen.

Reversible inhibition of rat hepatocyte proliferation by hydrocortisone and its effect on cell cycle-dependent hepatocarcinogenesis by N-methyl-N-nitrosourea.

The susceptibility of hepatocytes to carcinogenesis in vivo may be influenced by the phase of the cell cycle at which carcinogen-induced damage is incurred. In order to better understand this relationship, hepatic cell proliferation in juvenile male Fischer 344 rats was charted following a two-thirds partial hepatectomy. For hepatocytes, two distinct waves of DNA synthesis occurred which were followed after 6 to 8 hr by waves of mitotic cell division. In contrast to this kinetic pattern, when hydrocortisone was given after the partial hepatectomy, the initial waves of DNA synthesis and mitosis by hepatocytes were each delayed by about 15 hr. In rats not given hydrocortisone, susceptibility to heptocarcinogenesis by N-methyl-N-nitrosourea was greatest at 20 hr after partial hepatectomy when the peak fraction of proliferating hepatocytes was in the S phase. By shifting the time of onset of DNA synthesis, the hydrocortisone treatments also shifted the time with greatest sensitivity to N-methyl-N-nitrosourea, with hepatocytes in late G1 or S again the most susceptible. Numerous tumors were also induced by N-methyl-N-nitrosourea in extrahepatic tissues, including intestine, Zymbal's gland, nervous system, kidneys, odontogenic tissues, and peritesticular mesothelium. The results illustrate the importance of cell proliferation in carcinogenesis and further point to the specific sensitivity of certain cell cycle phases.

Effects of calcium and magnesium acetates on the carcinogenicity of cadmium chloride in Wistar rats.

The effects of calcium and magnesium acetates on the formation of injection site and testicular tumors in male Wistar rats over 2 years following s.c. injections of cadmium chloride (CdCl2) were determined. The rats (25/group) received a single s.c. dose of CdCl2 (0.02 or 0.04 mmol/kg; 0.9% NaCl solutions). Calcium and magnesium acetates were administered as 3% dietary supplements for 2 weeks prior to and 2 weeks after the CdCl2 injection, or as three daily s.c. injections (0.16 mmol calcium acetate per kg, 4 mmol magnesium acetate per kg; 0.9% NaCl solutions) at the same site as CdCl2 on the day before, the day of, and the day after CdCl2 dosing. Control groups were given 0.9% NaCl solution instead of CdCl2 plus s.c. or dietary calcium and magnesium acetates. In rats given injections of CdCl2 alone, the final tumor yields were 33 and 34% of rats at risk at the injection site (mostly fibrosarcomas) and 86 and 85% of rats at risk in the testes (mostly interstitial cell tumors), respectively, for the low- and high-CdCl2 doses. In control rats, the corresponding tumor yields were 0% at the site of 0.9% NaCl solution injection and 30% in the testes. Dietary calcium and magnesium acetates or s.c. calcium acetate did not affect significantly the tumor yields and latent periods. Simultaneous injections of magnesium acetate at the same site completely prevented the development of injection site tumors for both CdCl2 doses but had no effect on the final yields of testicular tumors. CdCl2 injection also caused significant elevation of incidence of the pancreatic islet cell tumors (8.5 versus 2.2%) regardless of any other experimental treatment. These results provide further evidence that the divalent carcinogenic metals may exert their activity through an antagonism with the physiologically essential divalent metals.

Histopathological evaluation of the inhibition of rat bladder carcinogenesis by 13-cis-retinoic acid.

An objective system for histopathological and statistical evaluation of rat bladder lesions induced by the carcinogen, N-methyl-N-nitrosourea, is described. This system has been used to measure the inhibitory effects of 13-cis-retinoic acid on the development of bladder cancer in female Wistar/Lewis rats. 13-cis-Retinoic acid caused significant inhibition of development of both preneoplastic and neoplastic lesions in bladder epithelium.

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.

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.

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)

Comparison of two-stage epidermal carcinogenesis initiated by 7,12-dimethylbenz(a)anthracene or N-methyl-N'-nitro-N-nitrosoguanidine in newborn and adult SENCAR and BALB/c mice.

In order to define factors which determine susceptibility to chemical carcinogenesis, mice sensitive (SENCAR) and resistant (BALB/c) to epidermal carcinogenesis were studied under several treatment conditions for sensitivity to initiation by 7,12-dimethylbenz(a)anthracene or N-methyl-N'-nitro-N-nitrosoguanidine and promotion by 12-O-tetradecanoylphorbol-13-acetate. In newborns of both strains, topical application of initiator was much less effective than in adults. However, initiation by i.p. injection of 7,12-dimethylbenz(a)anthracene is at least as effective in newborns as in adults, which may indicate that topically applied carcinogen is not delivered effectively to target cells in newborns. Thus, newborn epidermis can respond to 7,12-dimethylbenz(a)anthracene as well as adult epidermis when the initiator is appropriately administered. SENCAR mice are much more sensitive than are BALB/c mice to both initiators, which suggests that enhanced metabolic activation of hydrocarbon carcinogens by SENCAR mice is unlikely to account for their sensitivity. Newborn male SENCAR's developed approximately 50% more papillomas than did females in all groups. BALB/c newborn mice developed so few tumors that a meaningful comparison of sensitivity of males and females could not be made. Thus, the increased sensitivity of SENCAR's was apparent regardless of route of administration of initiator or the age or sex of the mice. SENCAR mice also developed a significant number of papillomas and squamous cell carcinomas with 12-O-tetradecanoylphorbol-13-acetate promotion in the absence of an exogenous initiator. Therefore, the skin of SENCAR mice may contain an initiated population of cells capable of responding to tumor promoters.

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.

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.

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.

Retinoic acid promotion of papilloma formation in mouse skin.

Retinoic acid is a weak promotor of skin tumorigenesis in Charles River CD-1 mice. Multiple papillomas were seen in 17% of the mice treated 3 times weekly with 5.1 micrograms retinoic acid for 20 weeks after initiation by a single treatment with 50 micrograms 7,12-dimethylbenz[alpha]anthracene (DMBA). These results suggest the necessity of a more thorough evaluation of retinoids as tumor promoters before their serious consideration as anti-cancer agents in man.

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.

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.