OS054. Differential strain sensitivities to ischemia/reperfusion in two mouse models of fetal growth restriction.

INTRODUCTION: Uterine ischemia/reperfusion (I/R) in rodents is a common model for the study of fetal growth restriction (FGR). It has been observed that different strains of mice vary in their response to uterine I/R. OBJECTIVES: Our objective was to characterize fetal and placental growth, and uterine and placental inflammation, in pregnant mice from 2 strains (C3H/HeOuJ and C57BL/6J), in response to different uterine I/R modalities. METHODS: Timed-pregnant mice (6-8 in each experimental group) were subjected to unilateral uterine I/R by either total flow restriction (TFR,right ovarian and uterine arterial clamping,5 or 30min) or partial flow restriction (PFR,right ovarian artery clamping,30min), or to sham-operation, on the 14th (for C57 mice) or 15th (for C3H mice) day of gestation. Four days later, fetal and placental weights and fetal loss were evaluated, and myeloperoxidase (MPO) activity was assayed in uterine and placental tissues. Data were analyzed by ANOVA with p<0.05 considered significant. RESULTS: In C3H/HeOuJ mice, TFR/30 min induced significantly (p<0.05) lower fetal and placental weights, and higher MPO activity in both uterus and placenta, compared to sham-operated controls. PFR/30min produced fetal but not placental growth restriction in the C3H mice. In contrast, C57BL/6J mice exhibited much greater sensitivity to uterine I/R: TFR/5 min was adequate to induce FGR in the C57 mice, which was equivalent in proportion to FGR caused by TFR/30min in C3H. C57 mice also exhibited significantly greater fetal loss and higher uterine, but not placental, MPO activity in response to I/R compared to sham-operated controls. CONCLUSION: Reliable FGR can be induced in different strains of mice by uterine I/R through adjustment of intensity, duration, and gestation day of the challenge. Mice of different strains have different tolerance to uterine I/R. These strain disparities would lend themselves to identifying the role of mouse strain-specific genetic determinants which form the bases for the observed differences in sensitivity to I/R-induced FGR.

Endothelin and the regulation of uteroplacental perfusion in nitric oxide synthase inhibition-induced fetal growth restriction.

The vasoactive mediators nitric oxide and endothelin are both produced in and active in the uterine and placental vasculature. Inhibition of nitric oxide synthase (NOS) results in fetal growth restriction. Endothelin (ET-1) is upregulated in the setting of NOS inhibition. Our purpose was to determine the impact of ET-1 on uterine and placental perfusion in the pregnant rat treated with a NOS inhibitor. Timed-pregnant Sprague-Dawley rats were treated with L-NAME (2.5 mg/kg/h), with and without A-127722 (10 mg/kg/day), or their respective vehicles, for 1, 4, or 7 days beginning on day 14 of gestation. Blood flow to various organs was determined by microsphere infusion. Maternal and fetal plasma nitrate/nitrite (NOx) was determined by fluorometric assay. Uterine and placental perfusion was significantly decreased by NOS inhibition and was restored to normal by ETA antagonism at 1 and 4 days of infusion but not at 7 days. Maternal plasma NOx, but not fetal plasma NOx, was significantly decreased by NOS inhibition alone. ETA antagonism in combination with NOS inhibition significantly lowered fetal plasma NOx. These results indicate that ET-1 is an important regulator of uterine and placental perfusion in the NOS inhibition model of fetal growth restriction. Our results also suggest that maternal administration of L-NAME does not result in significant transport of L-NAME across the placenta, but that addition of an ETA antagonist results in increased placental perfusion, allowing L-NAME greater access to the fetal compartment.

Influence of ET(B) receptor antagonism on pregnancy outcome in rats.

OBJECTIVE: To determine the effect of endothelin-B (ET(B))-selective receptor antagonism on pregnancy outcome in normal rats. METHODS: ET(B) receptor antagonist (A-192621; 5.0, 10.0, and 15.0 mg/kg per day) or vehicle was infused subcutaneously for 7 days by osmotic pump. Infusion was begun on day 14 of a 22-day gestation. Nonpregnant animals were treated similarly, and blood pressure (BP) responses and plasma antagonist levels were compared to those in pregnant animals. Mean arterial pressure (MAP) was measured on days 1, 4, and 7 of the infusion. Plasma ET(B) antagonist levels were measured on day 7 of infusion. On gestational day 21, fetal and placental weights and viability were evaluated at hysterotomy. Data were analyzed by analysis of variance and are presented as mean +/- standard error of the mean. RESULTS: Fetal and placental weights were significantly lower at doses of 10 and 15 mg/kg per day of the ET(B) antagonist compared with vehicle-treated controls (P <.001); these effects were less severe at 15 than at 10 mg/kg per day despite a fourfold higher plasma level of antagonist. Mean arterial pressure was significantly higher at 10 and 15 mg/kg per day compared with controls, but only on infusion day 1 (P <.05). In contrast, MAPs for nonpregnant rats were elevated throughout the infusion at all doses of the ET(B) antagonist (P <.05). CONCLUSIONS: ET(B) receptor antagonism inhibited fetal growth and increased maternal MAP in a dose-dependent manner, although the effect on BP was not sustained in pregnant animals. ET(B) receptor antagonism is detrimental to pregnancy outcome in the rat.

Focal nodular hyperplasia: CT findings with emphasis on multiphasic helical CT in 78 patients.

PURPOSE: To evaluate features of focal nodular hyperplasia (FNH) at multiphasic helical computed tomography (CT). MATERIALS AND METHODS: Clinical, pathologic, and preoperative imaging findings were retrospectively reviewed in 78 patients. Conventional liver CT was performed in nine patients; helical multiphasic CT, in 69. Diagnosis was based on complete resection (n = 20), biopsy (n = 42), or clinical and imaging follow-up for a minimum of 6 months (n = 16). Number, size, location, margins, surface, homogeneity of enhancement, and presence of a central scar, mass effect, exophytic growth, calcification, pseudocapsule, or vessels feeding or draining the lesion were evaluated. RESULTS: CT depicted 124 tumors (mean diameter, 4.1 cm; range, 1-11 cm); 62 were small (< or =3 cm). FNHs were hypervascular and hyperattenuating to liver on 106 of 106 arterial phase scans and were isoattenuating to liver on 82 of 89 delayed scans. Of the 124 tumors, 111 enhanced homogeneously, 109 had a smooth surface, 101 were subcapsular, 89 had ill-defined margins, and 62 had a central scar that was observed more often in large lesions (40 of 62 lesions) than in small lesions (22 of 62 lesions). FNHs less frequently exerted a mass effect (43 lesions), had vessels around or within the lesion (42 lesions), demonstrated exophytic growth (40 lesions), or showed a pseudocapsule (10 lesions). Only one FNH had calcification. CONCLUSION: Helical CT demonstrates characteristic features that may allow confident diagnosis of FNH. In typical cases, neither biopsy nor further imaging is necessary.

Differential effects of endothelin A and B receptor antagonism on fetal growth in normal and nitric oxide-deficient rats.

OBJECTIVE: To determine the role of endothelin (ET) in fetal and placental growth in rats with and without long-term nitric oxide synthase (NOS) inhibition. METHODS: Pregnant rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME) or saline and with one of three ET receptor antagonists or vehicle. The antagonists included A-182086 (nonselective) as well as A-127722 and FR-139317 (both ET(A) selective). Treatment was begun on day 14 of gestation. On gestational day 21, a hysterotomy was done. Litter size was recorded, and viability and fetal and placental weights were determined. Results were analyzed by analysis of variance or by a Kruskal-Wallis nonparametric analysis. RESULTS: In the absence of L-NAME, fetal and placental weights were not affected by ET(A)-selective antagonism but were significantly decreased by nonselective receptor antagonism (P <.001 and P <.05 for fetal and placental weights, respectively). Infusion of L-NAME resulted in fetal and placental growth restriction (P <.001). In the setting of L-NAME infusion, fetal and placental weights were increased by the ET(A)-selective antagonists (P <.01) but not by the nonselective antagonist, compared with weights from animals treated with L-NAME alone. There were more fetal deaths with L-NAME treatment (P <.05), but their occurrence was not significantly affected by any of the ET receptor antagonists. CONCLUSIONS: Endothelin-A antagonism alone did not affect fetal or placental growth, whereas combined ET(A) plus ET(B) antagonism produced fetal and placental growth restriction. In the setting of long-term NOS inhibition, ET(A)-selective antagonism improved fetal and placental growth, whereas antagonism of both ET(A) and ET(B) receptors did not. Endothelin contributes to NOS inhibition-induced growth restriction acting through the ET(A) receptor.

Endothelin and blood pressure regulation in the female rat: studies in normal pregnancy and with nitric oxide synthase inhibition-induced hypertension.

OBJECTIVE: To evaluate the role of endothelin (ET) in blood pressure regulation in normal pregnant and nonpregnant rats and with nitric oxide synthase (NOS) inhibition. METHODS: Pregnant and nonpregnant Sprague-Dawley rats were treated for 7 days with an ET(A)-selective (A-127722 or FR-139317), ET(B)-selective (A-192621), or ET(A)/ET(B) nonselective (A-182086) endothelin receptor antagonist, and/or with the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME, 2. 5 mg/kg/h). In pregnant rats, the ET antagonists and L-NAME were administered from gestational day 14 through day 21 (term = 22 days). All rats received indwelling arterial catheters for blood pressure measurement. Mean arterial pressures were recorded on infusion days 1, 4, and 7 and these data were compared by analysis of variance among experimental groups with p < 0.05 considered significant. RESULTS: The ET(A) receptor antagonism lowered blood pressure in both pregnant and nonpregnant rats (p < 0.05), whereas ET(B) antagonism resulted in hypertension (p < 0.001). ET(B) antagonism-induced hypertension was attenuated by pregnancy (p < 0. 001). Hypertension was induced in all rats treated with L-NAME (p < 0.001). Endothelin receptor antagonism, regardless of specificity, did not ameliorate L-NAME-induced hypertension in pregnant or nonpregnant female rats. The only observed effect of ET(A) antagonism on NOS inhibition-induced hypertension was the prevention of a continued rise at infusion day 7 in nonpregnant rats. CONCLUSIONS: Endothelin, acting via both the ET(A) and ET(B) receptors, contributes to blood pressure homeostasis in pregnant and nonpregnant rats. Endothelin receptor antagonism does not ameliorate NOS-inhibition-induced hypertension in pregnant rats.

Endothelin-1-induced placental and fetal growth restriction in the rat.

The objective of this study was to evaluate the effect of increased circulating endothelin on fetal and placental growth in the rat. Indwelling arterial and venous catheters were placed on day 14 of a 22-day gestation in timed-pregnant Sprague-Dawley rats. Saline, 0.2 nmol/kg/h endothelin, or 0.5 nmol/kg/h endothelin was continuously infused from day 15 through 21 in randomly assigned animals (n = 12 in each group). Maternal arterial blood pressure and serum endothelin levels were evaluated on days 15, 18, and 21 of gestation. On day 21, pregnancy outcome data including fetal and placental weights, litter size, and the occurrence of stillbirths were ascertained, and fetal blood was obtained for serum endothelin levels. All data were compared among the three groups, and statistical significance was determined by analysis of variance. Endothelin infusion resulted in a dose-dependent decrease in fetal and placental weights when compared to saline-treated pregnancies. A significant increase in maternal arterial blood pressure was noted only in the 0.5 nmol/kg/h endothelin group. Fetal and placental growth restriction occurred in the absence of maternal hypertension in the 0.2 nmol/kg/h group. These results demonstrate that endothelin infusion causes restriction of fetal and placental growth, even in the absence of maternal hypertension.

Endothelin receptor A antagonism prevents hypoxia-induced intrauterine growth restriction in the rat.

OBJECTIVE: Our purpose was to investigate the hypothesis that endothelin plays a critical role in maternal hypoxia-induced intrauterine growth restriction. STUDY DESIGN: Chronic indwelling venous and arterial catheters were placed on day 17 of a 22-day gestation in timed-pregnant Sprague-Dawley rats. Twelve rats were infused with saline solution and 12 with 6 mg/kg per day FR139317, an endothelin receptor A-specific antagonist. For gestational days 18 to 21 half the rats in each infusion group were housed in a normoxic environment and the other half in a hypoxic (14% oxygen) environment. On day 21 an arterial blood gas level was determined, the rats were then anesthetized, and a hysterotomy was performed. The weight of each pup and its corresponding placenta was recorded. Statistical significance was determined by analysis of variance. RESULTS: Among the rats receiving saline solution infusions, fetal weights were 20% less and placental weights were 11% less for those housed in a hypoxic environment compared with those housed in a normoxic environment (p < 0.003). Among the rats receiving FR139317 infusions, fetal and placental weights were not significantly different for those in a hypoxic environment compared with those in a normoxic environment. The fetal and placental weights for the rats receiving FR139317 infusion in hypoxic or normoxic environments were similar to those receiving saline solution in a normoxic environment. CONCLUSIONS: Endothelin plays a critical role in hypoxia-induced intrauterine growth restriction. Infusion of an endothelin antagonist prevents the intrauterine growth restriction caused by chronic hypoxia.

Resistance of murine lung tumors to xenobiotic-induced cytotoxicity.

Studies were performed to test the hypothesis that urethane-induced murine lung tumors exhibit xenobiotic resistance and alterations in pulmonary cytochrome P-450 enzymes. 1,1-Dichloroethylene, naphthalene, and paraquat were administered to tumor-bearing and control mice to elicit acute lung cytotoxicity, and responses were evaluated in tumors (papillary and solid), uninvolved surrounding tissue, and untreated control lung. 1,1-Dichloroethylene (125 mg/kg, i.p.) and naphthalene (225 mg/kg, i.p.) caused preferential necrosis of Clara cells in control lungs and uninvolved tissue of tumor-bearing lungs. In contrast, papillary and solid tumors were both resistant to 1,1-dichloroethylene-induced cytotoxicity. Paraquat (10, 20 mg/kg, i.v.) elicited Clara cell damage in control lungs and uninvolved lung tissue of tumor-bearing mice, with minor disruption of the alveolar epithelium. Neither papillary nor solid tumors sustained any apparent cell damage from paraquat. Immunoblots of P-450 enzymes confirmed constitutive expression of CYP2B1 in control lung and uninvolved lung tissue of tumor-bearing mice, but this P-450 enzyme was not detected in either adenomas or carcinomas. Lung CYP1A1 was inducible by beta-naphthoflavone in non-tumor-bearing mice and uninvolved tissue of tumor-bearing mice; however, inducibility was decreased in adenomas and abolished in carcinomas. These results demonstrate resistance of lung tumor cells to chemically induced cytotoxicity and diminished expression of cytochrome P-450 enzymes in tumors.

Lung adenoma structure among inbred strains of mice: the pulmonary adenoma histologic type (Pah) genes.

Urethane-induced, lung adenoma multiplicity and histologic type vary among mouse strains. We asked whether the Pas genes which control multiplicity also determine adenoma structure. Lung adenomas from inbred mice, F1 hybrids, and recombinant inbred mice were classified by growth pattern as either solid or papillary. Since no correlation was observed between adenoma multiplicity and histologic type, no linkage apparently exists between the Pas genes and adenoma morphology. We propose the name Pah (Pulmonary Adenoma Histologic type) for the genes controlling lung adenoma growth patterns. Genetic analysis indicated dominance of the papillary phenotype, and that two or more Pah genes determine adenoma structure.

NAD(P)H:quinone oxidoreductase (DT-diaphorase) activity and mRNA content in normal and neoplastic mouse lung epithelia.

DT-diaphorase (DTD) is a flavoprotein which catalyzes obligate two-electron reduction of a diverse group of substrates. We have reported previously that non-tumorigenic mouse lung alveolar type-II pneumocytes have high DTD activity, while cell lines derived from lung tumors do not. In contrast, other investigators, using human lung tissue, reported increased DTD activity in tumors compared with normal tissue. We therefore investigated DTD associated with mouse lung neoplasia in vivo as well as in vitro. Pulmonary tumors had far less DTD activity compared with normal mouse lung. Correspondingly, a tumorigenic mouse lung cell line which arose as a spontaneous transformant of a normal cell line had very low DTD activity compared with non-tumorigenic lung cells. DTD-specific mRNA levels were also much higher in normal cell lines than in neoplastic ones. DTD was localized histochemically in type-II pneumocytes in situ, but was not observed by this technique in normal bronchiolar epithelia or in tumor cells. These data show that, unlike what has been observed in human lung cancer, a marked decrease in DTD content and activity accompanied mouse lung tumorigenesis in vivo and neoplastic transformation in vitro.

Cells of origin of primary pulmonary neoplasms in mice: morphologic and histochemical studies.

Urethane-induced pulmonary adenomas in mice have two distinct histologic growth patterns--solid and papillary. The development of these tumors between 14 and 56 weeks was investigated in A/J mice. Solid tumor multiplicity remained constant from 14 to 56 weeks, whereas papillary and total tumor multiplicities increased in parallel between 14 and 28 weeks and remained constant through 56 weeks. The simplest explanation of these results is that solid and papillary adenomas arise independently, possibly from different cell types. The cell type of origin of these primary mouse lung tumors was investigated histochemically. Succinate dehydrogenase (SDH) histochemistry readily stained bronchiolar epithelial cells, but alveolar epithelial cells exhibited only slight enzymatic activity. Urethane-induced papillary adenomas exhibited intense SDH staining, whereas solid adenomas stained very lightly. Since Clara cells and type II pneumocytes are the only cells capable of proliferation in the bronchiolar and alveolar epithelia, respectively, the relative SDH activities of these adenomas is consistent with a hypothesis that solid tumors arise from type II pneumocytes and papillary tumors arise from Clara cells.

Differential staining of normal and neoplastic mouse lung epithelia by succinate dehydrogenase histochemistry.

The cell type of origin of primary mouse lung tumors was investigated histochemically. Succinate dehydrogenase (SDH) histochemistry readily differentiated bronchiolar epithelial (Clara and ciliated) cells from alveolar type II pneumocytes in mice aged 10 days through adult. Correspondingly, freshly-isolated Clara cells stained intensely but type II cells did not. Urethane-induced papillary adenomas exhibited intense SDH staining while alveolar adenomas stained very lightly. The relative SDH activities of these two types of pulmonary adenomas is consistent with the hypothesis that alveolar tumors arise from type II pneumocytes and papillary tumors from Clara cells.

Genetic analysis of the distribution of corticosterone-containing cells in mouse adrenal cortex.

Strains A/J and C57BL/6J (B6) differ in susceptibility to many neoplasms and infectious agents, with B6 mice generally being more resistant. Glucocorticoids protect against some of these pathologies. We examined the distribution of adrenocortical corticosterone (CS), the major endogenous glucocorticoid in mice, in these strains, using anti-CS serum. A distinct strain difference was found. B6 adrenals exhibited abundant CS-positive cells in cord-like arrays while A/J adrenals contained fewer, randomly arranged CS-positive cells. To quantify these results, each adrenal cortex was divided into eight sectors and each sector was classified as to phenotype. Ninety-three percent of the sectors of B6 cortices exhibited the cord-like pattern, whereas only 15% of the sectors of A/J cortices exhibited this pattern. These differences are consistent with a hypothesis that A/J mice are relatively deficient in the prophylactic activities of endogenous glucocorticoids. Adrenal glands from (C57BL/6J x A/J)F1 hybrid mice had approximately equal proportions of areas exhibiting each phenotype, indicating codominant alleles for this trait. We propose the name Cor for this gene. Thirty AXB and BXA recombinant inbred (RI) lines of mice derived from A/J and B6 progenitors were examined for CS immunostaining. Twenty-eight of them had either predominantly A/J-like or predominantly B6-like phenotypes. These RI data support either of two hypotheses. Hypothesis 1 emphasizes the nearly complete concordance of the RI lines with progenitor phenotypes and proposes that a single Cor gene regulates the distribution of CS-positive cells. Using this model, the strain distribution among RI lines implies linkage of Cor to a region on chromosome 6, 27-37 cM from the centromere. Hypothesis 2, which gives greater weight to the two RI lines with intermediate numbers of CS-positive cells, postulates an epistatic interaction between two Cor loci.

Evidence for a role of tert-butyl hydroxylation in the induction of pneumotoxicity in mice by butylated hydroxytoluene.

Previous studies have shown that BHT must be biotransformed, probably to a quinone methide, in order to cause pneumotoxicity in mice. When BHT is incubated with mouse hepatic or pulmonary microsomes, a major metabolite that is formed is the tert-butyl-hydroxylated derivative of BHT (BHT-BuOH). Herein we show that BHT-BuOH has a fourfold greater potency than BHT in increasing the lung wt/body wt ratio, decreases lung cytosolic Ca2+-dependent protease activity at 1/10 the dose required for BHT to do this, and causes pulmonary histopathology at 1/20 the dose of BHT. Lung damage occurs earlier and is repaired faster at lower concentrations of BHT-BuOH than of BHT, but the nature of the damage (type I cell death) and regenerative response (type II cell hyperplasia and differentiation) is apparently identical. Neither BHT-BuOH nor BHT cause damage to liver, kidney, or heart as assessed by light microscopy, so they are both specific pulmonary toxicants. We postulate that BHT-BuOH formation is an essential step in the conversion of BHT to the ultimate pneumotoxin, which might be the corresponding quinone methide.

Proliferating cell nuclear antigen (PCNA/cyclin) immunocytochemistry as a labeling index in mouse lung tissues.

Proliferating cell nuclear antigen is expressed in cells from late G1 through the S-phase of the cell cycle. Therefore, antibodies directed against this molecule should provide a probe for labeling immunocytochemically the nuclei of proliferating cells. Herein we demonstrate the feasibility and reliability of this technique by quantifying immunostained pulmonary nuclei. We applied polyclonal and monoclonal antisera to alveolar and bronchiolar pulmonary epithelial cells in various proliferative states in tissue-sections and in vitro. A/J mice had a slightly higher labeling index than C57BL/6J mice, and proliferation in both strains increased dramatically after butylated hydroxytoluene treatment produced compensatory hyperplasia of Type-II pneumocytes. Immunostaining in fetal and neonatal lung samples from mice was higher than in adults. Spontaneous lung adenomas had a higher labeling index than the surrounding normal lung tissue. In addition, new data contained herein demonstrate a strain difference in proliferation of bronchiolar epithelial cells, and quantify the extent to which BHT-induced lung damage increases these proliferative rates. This mammalian nuclear antigen did not cross-react with antiserum to a functionally related bacterial protein, the beta subunit of E. coli DNA polymerase-III holoenzyme.

Effects of adrenalectomy and corticosterone administration on mouse lung tumor susceptibility and histogenesis.

The effects of adrenalectomy (Ax) on urethan-induced lung tumors were determined in strains of mice that vary in their respective tumor susceptibilities: A/J (sensitive), BALB/cByJ (intermediate), and C57BL/6J (B6, resistant). Ax increased tumor number in both A/J (by 25%) and B6 mice (by 400%), but not in BALB/cByJ mice. The relative proportions of adenomas exhibiting the alveolar or papillary histological growth patterns were unchanged. Implantation of corticosterone-containing pellets into adrenalectomized B6 mice restored tumor multiplicity to that of sham-operated mice and into adrenalectomized A/J mice reduced multiplicity below that of sham-operated mice. Corticosterone, therefore, regulates neoplastic development of mouse lung epithelial cells.

Cellular derivation of lung tumors in sensitive and resistant strains of mice: results at 28 and 56 weeks after urethan treatment.

Urethan (CAS: 51-79-6)-induced pulmonary adenomas that arise from either alveolar type II pneumocytes of bronchiolar Clara cells have distinct histologic growth patterns and can thus be distinguished from each other. Strain differences were reported in the relative proportions of tumors derived from each cell type when these tumors were examined 14 weeks after urethan treatment. For determination as to whether these proportions changed at later stages of growth, tumors in A/J, SWR/J, RIIIS/J, BALB/cByJ, 129/J, and C57BL/6J mice were examined at 28 and 56 weeks after urethan treatment. Tumor multiplicity increased with time in all strains. Small tumors were predominantly type II cell derived in most strains, whereas medium and large tumors were derived mainly from Clara cells. This suggests that type II tumors are restricted in growth while Clara tumors may continue to grow. Medium and large Clara-derived tumors made up a larger proportion of the total tumor population at 28 and 56 weeks than at 14 weeks post urethan, even in A/J mice that typically display 85% type II cell-derived tumors at the earlier time. Several large Clara cell-derived tumors exhibited characteristics of cancer, whereas no type II cell-derived tumor was observed to do so. These results implicate the bronchiolar Clara cell as the predominant cell of origin of pulmonary adenocarcinomas in mice.

Genetic variation in the proliferation of murine pulmonary type II cells: basal rates and alterations following urethan treatment.

Susceptibility to urethan-induced pulmonary tumorigenesis varies among inbred strains of mice. A genetic basis for this variation was sought using three strains with widely differing tumor multiplicities after urethan treatment. Twenty-one mice from each of strains A/J (high susceptibility), BALB/cByJ (intermediate susceptibility; hereafter called cBy), and C57BL/6J (low susceptibility; hereafter called B6) were treated i.p. with 1 mg urethan/g body weight, and sacrificed at 0 (no urethan), 12, 24, 36, 48, 65, and 80 days after treatment (three mice per strain per time point). Each mouse was given 1 muCi [3H]thymidine/g body weight 45 min before sacrifice. Lungs were processed for autoradiography, and labeling indices were independently determined for non-tumor-associated type II cells and for tumor cells (most tumors arise from alveolar type II pneumocytes in A/J mice). Three categories of proliferative differences were found. First, statistically significant differences (P less than 0.05) among all strains were found for type II cell labeling indices in untreated mice, and these differences persisted for 65 days after urethan treatment. Proliferative rates were highest in A/J mice and lowest in B6 mice, while cBy mice were intermediate. Secondly, the peak of type II cell labeling occurred 12 days following urethan in strains A/J and cBy, but at 24 days in B6 mice. This difference is consistent with the fact that tumors were observed earlier following urethan treatment in A/J and cBy mice (at 36 days) than in B6 mice (at 48 days). Finally, the labeling indices in A/J and B6 tumors were high at first (6 and 4%) and then declined to 1-1.5% by 80 days after urethan treatment, while cBy tumor labeling indices remained at about 1.5% throughout the experimental period. These results suggest that the variation in susceptibility to urethan-induced lung tumorigenesis among different strains of mice is related to the normal basal rates of lung mitoses in these strains. Mice may be particularly sensitive to urethan during cell division, making strains with a higher rate of mitosis more susceptible to tumorigenesis.

The immunocytochemical demonstration of copper-zinc superoxide dismutase in the brain.

Copper-zinc superoxide dismutase (CuZn-SOD) has been localized in formalin-fixed, paraffin-embedded sections of both canine and rat brains. Staining with an immunoenzyme bridge sequence revealed CuZn-SOD in all regions of the brains examined. Specific sites of localization included cerebral cortical pyramidal cells, cerebellar Purkinje cells, neurons in 'subcortical nuclei', and oligodendrocytes throughout the brain. Similar sites of CuZn-SOD localization were identified in both species. These results are compared with reports by various investigators of SOD bioactivity in the brain.