It is now well established that nitric oxide (NO) is both a critical bioregulatory agent essential to normal physiological function and a potentially toxic species. The primary goal of our NO research program is to investigate the possibility that exposure to NO might increase cancer risk. After reviewing some initial chemical and cella culture experiments from our laboratories suggesting that this might be the case, we report on two in vivo studies using standard carcinogenesis bioassay protocols of potential relevance. In one, application of nitroglycerin and two other NO-donor drugs at the maximum tolerated dose to the skin of SENECAR mice followed by promotion with 12-tetradecanoylphorbol-13-acetate failed to reveal any tumor-initiating activity. In the second, treatment of strain A mice with two other compounds containing the NO-releasing N2O−2 functional group (by gavage in adults and intraperitoneal injection neonates) increased lung adenoma incidence only when the NO donor producing a potenitally carcinogenic alkylating agent as a by-product was employed. We conclude that the practical significance of NO exposure in term of human cancer risk remains to be established.

The tobacco-specific nitrosamine, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent carcinogen in adult rodents and variably effective transplacentally, depending on species. In pursuit of the thesis that human infants may be especially vulnerable targets for tumor initiation by tobacco smoke constituents, we tested the efficacy of NNK as a tumor initiator in infant mice. Cr: NIH(S) (NIH Swiss outbred) mice were given 50 mg/kg NNK i.p. on postnatal days 1, 4, 7, 10 and 14, with saline to controls. At an average age of 13 – 15 months, 57% of the NNK-exposed male offspring had hepatocellular tumors, with a multiplicity of 1.15 ± 1.4, including 4 with carcinoma. Liver tumors including 2 carcinomas were found in 8 (14%) of the NNK-exposed female offspring. There were no hepatocellular neoplasms in any control. A significant increase in primary lung tumors also occurred in the NNK-treated males, with an incidence of 3055 (57%) and a multiplicity of 0.7 ± 0.2, vs. 733 (21%), multiplicity 0.3 ± 0.6, in controls (P < 0.025). An apparent increase in the incidence of lung tumors in NNK-treated females, 2157 (37%) vs. 732 (22%) in controls, approached significance (P < 0.1). Thus NNK was a moderately potent neonatal carcinogen for liver and lung in infant Swiss mice and more efficacious in this regard than when received transplacentally by mice of the same strain.

The cytostatic drug procarbazine has previously been shown to be a potent transplacental neurotropic carcinogen in rats. Following a single IP administration of (14C-methylprocarbazine (110 mg/kg) on day 22 of gestation, methylation products with cellular DNA were determined in fetal and maternal rat organs. The concentration of the major adduct N7-methylguanine was highest in the maternal liver (224 μmol/mol guanine). Fetal and nonhepatic maternal tissues exhibited significantly lower levels, but differed little from each other. In brain, lung, intestines, and placenta the O6-methylguanine/N7-methylguanine ratio was close to 0.11, indicating that procarbazine, like other methylating carcinogens, initiates malignant transformation via methyldiazonium hydroxide as the ultimate reactant. Following a single dose of (14C-methyl)procarbazine to newborn animals, methylpurine values were 30–60 times lower than after prenatal administration. This suggests that DNA alkylation in nonhepatic tissues occurs by systemic distribution of a proximate carcinogen formed in the adult rat liver.

The developmental stages of 12 Erythrocebus patas embryos, ranging in gestational age from 30 to 50 days, is described. The pattern of embryogenesis in E. patas closely parallels the anatomic characteristics of human and other nonhuman primate embryos between stages 12 and 23. However, there is a delay in development in E. patas similar to that observed in human embryos which differs from the macaques and baboons. This temporal difference in the embryonic period is an important factor in the design and analysis of early pregnancy studies in this species.

Reproductive statistics were gathered over a 5½-year period on a colony of Erythrocebus patas. Pregnancies occurred throughout the year under laboratory conditions with a suggestion of a mating peak in the late fall and early winter. Menstrual cycles were monitored and found to average 30.6 days in length. Maximal vaginal cornification occured on day 15 of the cycle suggesting a midcycle ovulation. However, production of timed-mated pregnancies indicated ovulation occurred earlier and that breeding on days 10, 11, and 12 after menstruation was more likely to result in pregnancy. The gestation length was found to average 167.2 days in 142 harem-bred females and 167.5 days in 11 timed-mated pregnancies. Sixty-two percent of all pregnancies resulted in live births; 28% of the conceptions terminated with in-utero death of the fetus. Stillborn infants were delivered in 9% of the pregnancies. Infant mortality during the first 6 months of life was 10.2%. Females raised in the colony conceived their first offspring at approximately 3 years of age and males were able to sire infants at 3 years and 8 months.

Five fatal cases of disseminated strongyloidiasis were identified in Erythrocebus patas caged singly or in groups of two to four in an indoor research facility. This is the first report of fatal hyperinfective strongyloides infection in a species other than great apes and man. Severe pulmonary hemorrhage, duodenitis, and proximal colitis with microscopically demonstrable larvae in affected tissues were the key necropsy findings. E. patas is an available, suitable model for the study of disseminated strongyloidiasis.

Caged patas monkeys were evaluated monthly to determine changes in the color of their hair during infancy, adolescence, pregnancy and lactation. From birth until 3 months of age the facial and anterior crown hairs were short, sparse, and completely black. The body fur was a fine, short, fawn-colored hair mixed with longer black hairs which produced a black-tipped effect. During the second 3 months of life the body fur and anterior crown fur became coarser, longer, and changed to a red-brown color. The facial hairs thickened and became longer, but remained totally black. A thin line of black hairs outlined the brow and temple. The black chin hairs were gradually replaced by white from 7 to 24 months of age, and the upper lip hairs changed from black to white during the second year of life. Color changes related to pregnancy and lactation were confined to the nosepatch, cheek, and browline hair. The nosepatch and cheek hair changed from black or grey to completely white, and the browline faded to the approximate color of the body fur. These changes began approximately at the end of the second trimester of pregnancy, maximized during the third month of lactation, began to darken 1 to 2 months later, and returned completely to the black, nonpregnant colors approximately 1 year postpartum. In one nonlactating female, the darkening was delayed until 500 days postpartum and in one female ovariectomized in the light color phase, the darkening was complete 200 days later. The cause of these changes is believed to be hormonal, resulting from altered endocrine function during maturation and pregnancy, which may alter melanocyte stimulating hormone activity.

A rapidly fatal neoplastic disease with histological and clinical features resembling gestational choriocarcinoma in humans has been observed in patas monkeys. Timed pregnant females were given ethylnitrosourea (ENU) intravenously at doses of 0.1 to 0.4 mmol/kg body weight, beginning on day 30 of gestation and continuing weekly for a total of 12 injections. Of 59 monkeys given ENU during pregnancy, four of 12 subjected to the highest dose and three of the remaining 47 given lower doses died of choriocarcinoma within six months of cessation of ENU exposure. Death was usually caused by exsanguinating haemorrhage. At necropsy, tumour deposits were always numerous in the lungs and were frequently observed in abdominal viscera. An obvious primary uterine tumour was never found, and only one small primary was detected grossly. Sub-endometrial masses of tumour cells were generally observed microscopically, invading the endometrial stroma and forming endovascular tumour deposits in the veins. Both uterine and extrauterine tumour deposits were highly haemorrhagic, often partially necrotic, and consisted of cytotrophoblast-like cells with frequent mitoses, a high degree of cellular pleomorphism and variable but often prominent cytoplasmic glycogen. This tumour was never seen in males or non-gravid adult females. Chorionic gonadotrophin assays conventionally used for human and macaque samples were negative in both normally pregnant and tumour-bearing patas, and did not contribute to the diagnosis. Trophoblast of patas monkeys appears highly susceptible to the carcinogenic effects of ENU and provides an animal model for gestational choriocarcinoma.

Administration of phenobarbital for 5 to 7.5 weeks to aged C3HfB/HeN mice with spontaneous liver tumors produced an enhancement of gamma glutamyl transpeptidase activity in the tumors and a decrease in glucose-6-phosphatase and adenosine triphosphatase activity. Discontinuation of phenobarbital feeding for 5.5 weeks resulted in the loss of gamma glutamyl transpeptidase activity in tumors. These findings of alterations in three membrane-associated enzymes indicate that phenobarbital produces substantial changes in the composition of cellular membranes which may be related to its promoting activity.

Spontaneous mouse liver nodules were found to be resistant to iron accumulation induced either by dietary overload or by a rapid protocol of subcutaneous injection of iron dextran. Transplants of 11 liver nodules into the mammary fat pad gave rise to neoplastic growth. Transplants of the less differentiated nodules grew more frequently and rapidly than better differentiated nodules and produced pulmonary metastases. Therefore, these mouse liver nodules are considered to be neoplasms, and their resistance to iron accumulation suggests that this marker will be useful in studying their histogenesis.

A single intraperitoneal dose of methyl(acetoxymethyl)nitrosamine (13 mg/kg body weight) given to 78 5-week-old male rats induced 25 mesotheliomas; two mesotheliomas were found in 67 control rats. All mesotheliomas arose from the peritesticular mesothelium and had a typical microscopic appearance of branching papillary fronds with a collagenous core covered by one or many layers of plump tumor cells. Cytoplasm of tumor cells contained material that reacted positively to a colloidal iron stain and was labile to hyaluronidase. In addition to frank mesotheliomas, 16 lesions, which we called atypical mesothelial proliferations. were found. These consisted of a single focus of plump mesothelial cells overlying an area of thick stroma. Often these foci included short, non-branched papillary projections above the surface of adjacent normal mesothelium. Twelve of the 16 lesions occurred in methyl(acetoxymethyl)nitrosamine-treated rats.

An extensive survey of markers for transformation of adult rat liver-derived epithelial-like cell lines was conducted. The cell surface properties associated with tumorigenicity were reported earlier. Now we report the correlation of various growth characteristics and enzyme activities with tumorigenicity. Among markers identified that correlated with tumorigenicity within the population, biochemical assay of γ-glutamyl transpeptidase activity was specific but not sensitive. Rapid growth rate accompanied by increased cell density showed a positive trend with tumorigenicity. Activity of plasminogen activator in cell lysates was not correlated with tumorigenicity. Among identified markers that gave an estimate of the fraction of tumorigenic cells in cultures, growth in soft agar was a good quantitative marker. Cytochemical activity of γ-glutamyl transpeptidase was a specific and sensitive marker. Colony-formation in low-calcium medium also distinguished tumorigenic cells from nontumorigenic ones. Colony-forming efficiency in both 10 and 1% serum media was somewhat higher for tumorigenic lines, but the difference from non-tumorigenic lines was not great enough to make this a useful marker. Thus, among 11 properties examined in both parts of this survey, biochemical assay of γ-glutamyl transpeptidase activity, high uptake of 2-deoxy-d-glucose, and growth pattern in liquid medium can serve as population markers, while growth in soft agar, cytochemical assay of γ-glutamyl transpeptidase activity, and growth in low-calcium medium can be used as cell markers. Combined use of these markers in adult rat liver lines provides a reliable means of identifying transformation in an epithelial cell system.

Male Sprague-Dawley (Charles River CD) rats received a single carcinogenic dose (12 mg/kg) of the α-acetoxy derivative of dimethylnitrosamine, N -[ 14 C]methyl- N -acetoxymethylnitrosamine, and were allowed to survive for 12 hr. Following i.v. injection, highest concentrations of 7-methylguanine and O 6 -methylguanine were present in DNA of the lung, the principal target organ in the carcinogenesis by N -methyl- N -acetoxymethylnitrosamine at this dosage by this route of application. Injection i.p. of a similar dose of N -[ 14 C]methyl- N -acetoxymethylnitrosamine led to preferential DNA alkylation in organs bordering the abdominal cavity, with highest levels of methylated purines in ileum and colon, the principal sites of tumorigenesis for this route of administration. Esterases potentially responsible for the bioactivation of N -methyl- N -acetoxymethylnitrosamine in vivo were found in all organs investigated, with the highest levels of activity being present in rat kidney and liver. Incubation of N -[ 14 C]methyl- N -acetoxymethylnitrosamine with DNA and esterases from rat kidney in vitro resulted in a pattern of methylated purines similar to that produced by N -methyl- N -nitrosourea and related methylating carcinogens, indicating that these agents, including dimethylnitrosamine, exert their biological effects through a common alkylating intermediate. Pretreatment of rat liver extracts with the esterase inhibitor diisopropyl fluorophosphate (10 −4 m) reduced both the decomposition of N -methyl- N -acetoxymethylnitrosamine and DNA alkylation in vitro by more than 90%.

A breeding colony of the Old World monkey Erythrocebus patas, an African species, has been established to study transplacental carcinogensis in a representative primate species. ENU was administered by repeated iv injections to pregnant females and to juveniles of both sexes. Repeated doses of 0.1 mmole/kg body weight per injection, given at 14-day intervals, are tolerated without apparent signs of toxicity by fetal and by pregnant and nonpregnant adult or juvenile monkeys. The internal can be reduced to 7 days, at least during the latter two-thirds of pregnancy. Large single doses (1.0 mmole/kg) are tolerated by pregnant females but are frequently abortifacient. These doses produce acute cytolytic damage to the cells of the periventricular germinal matrix in the fetal brain. Studies with [14C]ethyl-ENU indicate that there is no placental barrier to this carcinogen. As of December 1975, no tumors had been observed.

Continuous epithelial-like cell lines derived from normal adult rat liver and hepatocarcinomas were evaluated for their growth in soft agar and five properties of the cell membrane as markers for neoplastic transformation. A correlation of these properties was made to the tumorigenicity of the lines in nude mice. Growth in soft agar was a specific and sensitive marker, whereas the data on uptake of 2-deoxy-D-glucose were consistent, with high uptake being a specific but clearly not a sensitive marker. Agglutination and hemadsorption mediated by concanavalin A, multinucleation in the presence of cytochalasin B, and the cell membrane activity of adenosine triphosphatase did not correlate with tumorigenicity of the other markers for transformation. In addition, it is shown that Mycoplasma infection does not alter any of these properties but that infection can be eliminated by passage of cells through nude mice.

Seven adult rat liver epithelial lines derived from normal livers and three hepatocarcinoma lines were examined for tumorigenicity in nude mice and the production of plasminogen activating factor (PAF). All normally-derived, nontumorigenic liver epithelial lines produced PAF while one tumorigenic hepatocarcinoma line did not. Thus, for these epithelial cells there is no absolute correlation between PAF production and tumorigenicity.

Cryptococcosis was diagnosed in an adult male patas monkey (Erythrocebus patas) 9 months after importation. The disease was characterized by an open lesion on the buttock and epileptiform seizures. Diagnosis was confirmed through immunologic identification of the organism in serum and cerebrospinal fluid and by mycologic procedures performed on the cultured organism. The monkey was killed and cryptococcal organisms were found in the lung, brain, subcutaneous lesions, thyroid, pancreas, adrenals, and spinal cord. Retrospective analysis of stored serum indicated that the monkey was infected at the time of importation.