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There has been considerable interest in the use of genetically modified mice for detecting potential environmental carcinogens. For this reason, the National Toxicology Program has been evaluating Tg.AC hemizygous and p53 haploinsufficient mice as models to detect potential carcinogens. It was reasoned that these mouse models might also prove more effective than standard rodent models in evaluating the numerous disinfection byproducts that are found in low concentrations in drinking water. Dichloroacetic acid (DCA) is one of the most frequently found disinfection byproducts and DCA has been consistently shown to cause hepatocellular tumors in rats and mice in standard rodent studies. Tg.AC hemizygous and p53 haploinsufficient mice were exposed in the drinking water to DCA for up to 41 weeks. In a second study Tg.AC mice were subjected to dermal DCA exposure for up to 39 weeks. Increased incidences and severity of cytoplasmic vacuolization of hepatocytes were seen in the p53 mice, but there was no evidence of carcinogenic activity at exposures of up to 2000 mg/l in the drinking water. Increased incidences and severity of cytoplasmic vacuolization of hepatocytes were seen in the drinking water study with Tg.AC mice and a modest non-dose-related increase in pulmonary adenomas was observed in males exposed to 1000 mg/l in the drinking water. Dermal exposure up to 500 mg/kg for 39 weeks resulted in increased dermal papillomas at the site of application in Tg.AC mice. No significant increase in papillomas under the same study conditions was seen in the 26-week study. For DCA under these study conditions, the p53 and Tg.AC mice appear less sensitive to hepatocarcinogenesis than standard rodent models. These results suggest caution for the use of Tg.AC and p53 mice to screen unknown chemicals in drinking water for potential carcinogenicity.

Tg.AC mice develop epidermal papillomas in response to treatment with dermally applied nongenotoxic and complete carcinogens. The persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a multi-site rodent carcinogen and tumor promoter that induces the formation of papillomas in Tg.AC mice. To examine the dose-response relationship and compare dermal and oral routes of exposure for TCDD-induced skin papillomas, female Tg.AC mice were exposed dermally to average daily doses of 0, 2.1, 7.3, 15, 33, 52, 71, 152, and 326 ng TCDD/kg/day or 0, 75, 321, and 893 ng TCDD/kg body weight by gavage for 26 weeks. The incidence of cutaneous papillomas was increased in a dose-dependent manner, and tumors developed earlier with higher exposure to TCDD regardless of route of administration. Increased incidences of cutaneous squamous cell carcinomas were observed in mice exposed to dermal (> or =52 ng/kg) and oral (893 ng/kg) TCDD. Higher gavage doses than dermal exposure doses were required to induce papillomas and squamous cell carcinomas. Despite a linear correlation between administered dose and terminal skin concentrations, the incidence of tumor formation was lower in the gavage study than in the dermal study with respect to mean terminal skin TCDD concentrations. These studies demonstrate that, although Tg.AC mice are less responsive to TCDD by gavage than by dermal exposure, the induction of skin neoplasms is a response to systemic exposure and not solely a local response at the site of dermal application. Differences in response between the routes of exposure may reflect pharmacokinetic differences in the delivery of TCDD to the skin over the duration of the study.

Elmiron (sodium pentosan polysulfate) is used for the relief of urinary bladder pain associated with interstitial cystitis. The National Toxicology Program (NTP) tested this compound because of its orphan drug status and lack of information about its chronic toxicity and carcinogenicity. Groups of 50 male and 50 female F344/N rats were given Elmiron in de-ionized water by gavage at doses of 0, 14, 42, or 126 mg/kg to males and 0, 28, 84, or 252 mg/kg to females once daily, 5 days per week, for up to 2 years. The same numbers of male and female B6C3F1 mice were dosed similarly with 0, 56, 168, or 504 mg/kg. Elmiron administration produced no effect on the body weight of rats, male mice, or low- and mid-dose groups of female mice. The body weights of the high-dose female mice were significantly decreased relative to those of controls. Pairwise comparison showed that survival of all dosed groups of rats and mice was similar to that of the controls. Elmiron was not carcinogenic in F344/N rats. An increased incidence of liver hemangiosarcoma provided evidence of some carcinogenic activity for Elmiron in male B6C3F1 mice. Increased incidences of liver hemangiosarcoma, hepatocellular neoplasms (predominantly adenomas), and malignant lymphomas revealed carcinogenic activity of Elmiron in female B6C3F1 mice. Elmiron administration produced elevated occurrences of nonneoplastic lesions, such as vacuolated histiocytes in the rectum, lung, spleen (males only), and mesenteric lymph node in rats and liver, rectum, mesenteric lymph node, and spleen in mice. Myxomatous change, chronic inflammation, and squamous metaplasia (mice only) were observed in the large intestine, and lymphohistiocytic hyperplasia was found to be increased in the spleen of rats of both sexes treated with the highest dose. In the latter lesion, the histiocytes contained pale, finely granular cytoplasm and were not considered to represent the same change as the vacuolated histiocytes seen in the mesenteric lymph node and rectum. Under the conditions of these 2-year studies, Elmiron was carcinogenic to mice but not rats.

Frankia are Gram-positive, filamentous bacteria capable of fixing atmospheric dinitrogen either in the free-living state or in symbiosis with a variety of woody plants. Only a few Frankia genes have been sequenced and gene expression is not well characterized. To isolate a segment of Frankia DNA that functions as an RNA polymerase promoter, fragments of Frankia strain ArI5 genomic DNA were cloned upstream of a promoterless, Vibrio harveyi luxAB cassette. Constructs were screened for luminescence in E. coli and positive clones assayed for in vitro transcription activity with a partially purified Frankia RNA polymerase extract. Primer extension analysis of in vitro transcripts produced from one clone, GLO7, identified two major transcription start sites, TSP-1 and TSP-2, 52 bp apart. Deletion analysis then localized sequences essential for promoter activity. The upstream promoter region, GLO7p1, contains sequences resembling the -35 element of a Streptomyces promoter and the -35 and -10 elements of the canonical E. coli promoter. Also within this region are two pentamers identical to sequences near the 5' end of the Frankia strain CpI1 glutamine synthetase gene. The second promoter, GLO7p2, contains a putative NtrC binding site at -145 and a possible sigma(N)-RNA polymerase recognition sequence at -14 suggesting that GLO7p2 may be a nitrogen-regulated promoter. An in vivo transcript representing an ORF of 498 aa starting 64 bp downstream of the distal transcription start, TSP-1, was detected by RT-PCR. This supports the conclusion that this DNA fragment has promoter activity in vivo as well as in vitro.