Marie C. LaRegina

A chemically induced model for squamous cell carcinoma of the lung in mice: histopathology and strain susceptibility.

Lung cancer, primarily associated with tobacco use, is the leading cause of cancer morbidity and mortality in the United States. Squamous cell carcinoma (SCC) is one of the four major histological types of lung cancer. Although there are several established models for lung adenoma and adenocarcinomas, there is no well-established mouse model for lung SCC. We treated eight different inbred strains of mice with N-nitroso-tris-chloroethylurea by skin painting and found that this regimen induced lung SCCs in five strains of mouse (SWR/J, NIH Swiss, A/J, BALB/cJ, and FVB/J) but not in the others (AKR/J, 129/svJ, and C57BL/6J). Mouse lung SCCs have similar histopathological features and keratin staining to human SCC. Moreover, a wide spectrum of abnormal lung squamous phenotypes including hyperplasia, metaplasia, carcinoma in situ, and invasive carcinoma, were observed. There are strain-specific differences in susceptibility to Lscc induction by N-nitroso-tris-chloroethylurea with NIH Swiss, A/J, and SWR/J mice developing scores of SCCs whereas the resistant strains AKR/J, 129/svJ, and C57BL/6J failed to develop any SCCs. FVB/J and BALB/cJ mice had an intermediate response. We conducted whole-genome linkage disequilibrium analysis in seven strains of mice, divided into three phenotype categories of susceptibility, using Fisher’s exact test applied to 6,128 markers in publically available databases. Three markers were found significantly associated with susceptibility to SCC with the P < 0.05. They were D1Mit169, D3Mit178, and D18Mit91. Interestingly, none of these sites overlap with the major susceptibility loci associated with lung adenoma/adenocarcinoma development in mice. The mouse SCC described here is highly significant for preclinical studies of lung cancer chemopreventive agents because most human trials have been conducted against precancerous lesions for SCC. Furthermore, this model can be used in determining genetic modifiers that contribute to susceptibility or resistance to lung SCC development.

Testicular Cytotoxicity of Intravenous Doxorubicin in Rats

Although the testicular cytotoxicity of many chemotherapeutic drugs has been evaluated in mice, their small size can pose technical problems. In this report, we describe doxorubicin-induced testicular toxicity in a larger animal model, the Sprague-Dawley rat. Fifty-three rats were used for this study. On day 0, rats in the treatment groups were anesthetized and given different single intravenous doses of doxorubicin (0.1 to 30 mg./kg.). On day 56 +/- 2, all surviving rats were killed and necropsied. Testicular toxicity was evaluated qualitatively by histology and quantitatively by testicular weight, sperm head count, repopulation index and epididymal index. The histologic effects of doxorubicin on the heart, liver and kidney were qualitatively evaluated. Progressive dose-dependent testicular atrophy and oligospermia occurred at low and intermediate dosages of doxorubicin (0.1 to 5 mg./kg.). Marked testicular atrophy, oligospermia and germinal aplasia were observed at high dosage of doxorubicin (10 mg./kg.). LD50 for animal mortality at day 56 +/- 2 for doxorubicin appears to be 10 mg./kg. These findings are similar to those reported in mice. The rat is a suitable model for the study of techniques to avoid drug-induced testicular damage.

Response to exogenous cholecystokinin of six human gastrointestinal cancers xenografted in nude mice

Gastrointestinal hormones regulate growth of cancers as well as normal tissues. We investigated whether long-term cholecystokinin (CCK) administration might affect growth or metabolism of human tumors xenografted in nude mice. In each experiment, approximately 20 nude mice bearing subcutaneous xenografts of the particular cancer line being studied were used. Half received CCK and half received saline solution intraperitoneally twice daily for 14 days. Tumor volume and body weight were measured every 3 days. If the tumors produced marker substances, these were measured in nude mouse serum and also in the xenografts. Tumor growth was significantly retarded by CCK in two of the six cancers studied. In each case, DNA, RNA, and protein reflected tumor volumes. In one of these tumors (SLU 077), serum carcinoembryonic antigen (CEA) levels paralleled the tumor volumes. In another tumor (SLU 132), serum CEA levels and tumor immunolabeling for CEA and pancreatic oncofetal antigen increased in response to CCK administration, whereas tumor volumes did not. These findings suggest that exogenous highdose CCK altered the growth and metabolism in two of six human cancers studied.

Regional doxorubicin delivery reduces testicular toxicity

Many anti-cancer drugs cause infertility. Regional delivery of these agents is a potential method to avoid this problem. We investigated the protective effect of normothermic testicular circulatory arrest on gonadal toxicity during doxorubicin administration in the Sprague-Dawley rat. Four groups of eight rats each were used. Animals in group 1 received no treatment. Rats in group 2 were anesthetized and received a bolus of intravenous doxorubicin (6 mg/kg). In groups 3 and 4, normothermic circulatory isolation of the left testis was induced by cross-clamping of the spermatic cord and gubernaculum immediately before doxorubicin administration. This was maintained for 15 min after doxorubicin administration in group 3 and for 45 min in group 4. Cessation and return of testicular blood flow were confirmed by Doppler. On Day 56, all rats were killed and necropsied. Testicular toxicity was evaluated qualitatively by histology and quantitatively by measurement of testicular weight, sperm count, repopulation index, and epididymal index. The results indicated that 15 min of testicular circulatory isolation mitigated testicular toxicity to a small extent and that 45 min of circulatory isolation provided moderate protection against doxorubicin-induced testicular toxicity.

Exogenous cholecystokinin (CCK) reduces neonatal rat brain opioid receptor density and CCK levels

Newborn rats were given saline or cholecystokinin8 (CCK8) (5 micrograms/kg, twice daily) i.p. for 3 weeks. On day 21, effects on brain development were assessed. CCK-like immunoreactivity was measured in 7 brain regions; a small (12-18%) but significant decrease in endogenous levels of this peptide was detected in cerebral cortex, medulla and pons of the CCK-treated rats. Morphometric measurements revealed a slight reduction in thickness of most cerebral cortical sections within the CCK-treated group. The area of a midsagittal section of the cerebellum was unchanged except for the Purkinje/granule cell layer, which was smaller in CCK-treated animals. Levels of mu-, delta- and kappa-opioid receptors were estimated by homologous displacement binding assays using selective radioligands. The CCK treatment resulted in a significant decrease in levels of mu- (11%) and delta- (13%)-sites in the cerebral cortex. Neither binding affinities nor kappa-receptor densities were altered. Other animals received the same treatment regimens for 21 days and were maintained for an additional 29 days without treatment; these rats had reductions only in cortical mu-sites (15%). Chronic intraventricular administration of CCK (0.1 microgram/h) to adult rats did not elicit a similar down-regulation of cortical mu or delta receptors, suggesting that the effects observed in neonates reflected developmental processes.

Enhanced susceptibility to chemical induction of ovarian tumors in mice with a germ line p53 mutation.

Mice with a germ line p53 mutation (p53Ala135Val/wt) display increased susceptibility to lung, skin, and colon carcinogenesis. Here, we show that p53Ala135Val/wt mice developed ovarian tumors significantly more rapidly than their wild-type littermates after 7,12-dimethylbenz(a)anthracene (DMBA) treatment. Approximately 50% of the ovarian tumors in p53wt/wt mice and 23% in p53Ala135Val/wt mice are adenocarcinomas and the remaining tumors were adenocarcinoma mixed with sarcoma or ovarian sarcomas. All of the p53Ala135Val/wt mice had died of ovarian tumors 25 weeks after the initial DMBA treatment, whereas >50% of p53wt/wt mice were still alive. These mice not only have a shortened tumor latency but also closely resemble a subset of human ovarian tumors containing the p53 mutation. Microarray and GenMAPP analyses revealed that the mutant p53 (Ala135Val) affected several cellular processes, including the cell cycle, apoptosis, and Wnt pathways. These findings indicate that a germ line p53 mutation significantly enhanced DMBA-induced ovarian tumor development and progression. (Mol Cancer Res 2008;6(1):99–109)

Effect of chronically administered somatostatin on the nude mouse

We investigated the impact of chronically administered synthetic cyclic somatostatin-14 on the nude mouse. Four groups of seven animals each received intraperitoneal injections twice daily for 14 days with diluent or 5, 50, or 500 mcg/kg somatostatin in hydrolyzed gelatin. Overall health and body weight were unaffected by this treatment. Among the seven organs examined at necropsy, kidney alone showed a dose-related gain in weight with increasing somatostatin dose. Renal RNA content also increased in response to somatostatin treatment, but liver DNA content and small bowel DNA content and protein content decreased. This work indicates that somatostatin affects metabolism of gastrointestinal tissues and provides evidence that renal growth can be regulated by somatostatin. Whether similar effects occur at a physiological somatostatin level is unknown, but the current findings suggest new therapeutic possibilities for this hormone.

Preservation of fertility following doxorubicin administration in the rat

Several hundred thousand men receive chemotherapy each year; many are sterilized by this treatment. Testicular circulatory isolation (TCI), a regional drug exclusion approach to circumvent chemotherapy-related infertility, lessens doxorubicin-induced testicular injury in the rat. We evaluated the effect of TCI on doxorubicin-induced infertility in this study. Thirty-two eight-week-old male Sprague-Dawley rats were used. Eight rats received TCI for 45 min. Eight received doxorubicin (i.v. bolus) plus sham surgery. Eight received i.v. doxorubicin given immediately after institution of TCI. Eight controls received sham surgery alone. Mating studies began 2 months later. Six of the 8 males receiving TCI alone were fertile. In the doxorubicin-treated, sham-operated group, 0 of 7 animals were fertile. In the doxorubicin-treated group which also received TCI, 2 of 7 males were fertile. In the sham-operated group, all 8 rats were fertile. This is the first evidence that regional drug exclusion technique can improve fertility in this model.

Effect of chronically administered cholecystokinin on the nude mouse

The nude mouse has been used to evaluate the effect of cholecystokinin (CCK) on xenografted tissues, but little is known about long-term actions of cholecystokinin on native organs in this animal. We investigated the impact of chronically administered synthetic cholecystokinin octapeptide on the nude mouse. Six groups of eight animals each received intraperitoneal injections twice daily for 14 days with diluent or a 4-log range of cholecystokinin. Overall health, behavior, and body weight were unaffected by this treatment. Among the seven organs examined at necropsy, pancreas alone showed a dose-related increase in weight. Pancreatic DNA content decreased with increasing dosages of CCK-8, while RNA content exhibited a biphasic response to CCK-8. The only histological abnormality occurred in the pancreas and was confined to the higher doses. These data indicate for the first time the action of CCK on the non-tumor-bearing nude mouse. Unlike other animal models, the nude mouse responds to cholecystokinin administration with pancreatic hypoplasia and hypertrophy, which is accompanied by pancreatitis at higher doses.

Proximal gastric vagotomy by minimally invasive methods in an acute rat model

SummaryIn this prospective study, minimally invasive methods of proximal gastric vagotomy (PGV) were investigated in male Sprague-Dawley rats. Completeness of vagotomy by traditional operative therapy, by laser denervation of the gastric serosa, and by subserosal or transmucosal injections of chemoneurolytic agents was evaluated with postoperative Congo red testing, ulcerogenic stimulation of the gastric mucosa, and histochemical labeling of whatever vagal fibers remained in the gastric wall. Short-term results demonstrate that successful PGV can be performed with minimally invasive methods.