Mutagenesis of a single AT basepair in mice transgenic for PhiX174 am3, cs70. II. Brain.

Most cell divisions in the mouse brain have ceased by 14 days after birth. Therefore, spontaneous mutations that occur in brain cells can be assumed to be fixed by replication during brain development. Spontaneous and ethylnitrosourea (ENU)-induced reverse mutations at a single AT base pair were measured in brain tissue by using mice transgenic for PhiX174 am3, cs70. The line (am54) has 50 PhiX genomes per haploid genome integrated in a tandem array and is maintained by random breeding on a C57BL/6 background. For mutagenesis studies, homozygous am54 males were mated to non-transgenic C57BL6/J females. Four-day old offspring from this cross were treated with 50 mg/kg ENU and were euthanized at 68-80 days of age. The ENU-treated animals had a significantly higher frequency of am3 revertants in brain than did concurrent controls. In a second experiment, hemizygous male offspring (85 to 94 days old) were treated with 150 mg/kg ENU and euthanized at various post-injection intervals (3, 10 and 110 days). The revertant frequencies 3 and 10 days after treatment were not significantly different from control values. At the 110 days post-injection interval, however, the average revertant frequency in the treated group was significantly lower than controls. In a second study animals were euthanized 3, 10 and 74 days after treatment. Two groups (3 and 74 days post-injection) also showed a significant decrease in the revertant frequency as compared to controls. Additional sets of adult animals were treated with 50 and 150 mg/kg ENU and were euthanized 195 to 201 days after treatment. The average revertant frequency of the animals that were treated with 50 or 150 mg/kg ENU was not significantly different from the control value. Thus, although an increase in mutant frequency is detected in the PhiX174 system when neonatal mice are treated with ENU, treatment of mature mice with ENU did not result in an increase in the mutant frequency. Moreover, under certain conditions, ENU-produced a significantly lower mutant frequency than was observed in the control animals. This decrease in the revertant frequency among the treated animals was likely due to selective killing of cells with a higher spontaneous revertant frequency than cells with lower frequency.

Mutagenesis of a single AT basepair in mice transgenic for PhiX174 am3 cs70 I. Spleen and testis.

Mutations induced in a single AT base pair were studied in spleen and testis by using mice transgenic for PhiX174 am3, cs70 and ethylnitrosourea (ENU) as the mutagen. The transgenic mice were produced on the C57BL6/J background. The line (am54), which carries 50 copies of PhiX per haploid genome integrated in a tandem array, was selected for experimental use and was maintained by random breeding. The animals for mutagenesis studies were produced by mating homozygous am54 males to wildtype C57BL6/J females. Hemizygous male offspring (8 to 10 weeks old) from this cross were injected i.p. with 150 mg ENU per kg and were euthanized 3, 10 or 110 days after treatment. The spontaneous revertant frequency in the spleen was 1.42 x 10(-6) per plaque forming unit (pfu) and in the testis it was 1.41 x 10(-6) per pfu. There was no significant difference between the two tissues. In spleen, it was not until 110 days after ENU treatment that the average revertant frequency among treated animals was significantly higher than the revertant frequency among the control animals. In spleen, the induced frequency of basepair substitutions in the center AT basepair in the am3 nonsense codon was 2 x 10(-6). Also at this post-injection interval the variance of revertant frequencies in the spleen was not different from control variance. In testis, the average revertant frequency 110 days post ENU injection was not significantly different from the control. However, two important observations were made regarding the testis data. First, one animal had a significantly increased revertant frequency 110 days after ENU treatment in comparison to the other four animals in the group that had revertant frequencies equal to or lower than the average control frequency. Second, the variance of revertant frequencies in the testis among the treated animals increased as the post injection period increased. Taken together, these observations may indicate that the revertants formed large clusters in one testis sample.

Relationship between clinical stage and histological zone of origin in early prostate cancer: morphometric analysis.

A detailed morphometric analysis of 96 radical prostatectomy specimens (13 clinical stage A1, 29 A2, 34 B1 and 20 B2) was undertaken to examine the relationship of zone of origin to volume, grade and extraprostatic extension of cancer. In patients with stage A disease, transition zone (TZ) cancer (present in 81%) was significantly larger but of lower grade than peripheral zone (PZ) cancer (present in 90%). The total volume of cancer in stage A1 patients averaged 1.55 ml with 72% of TZ origin. In patients with stage A2 disease, tumour volume averaged 5.83 ml with only 57% of TZ origin. Specimens taken during transurethral resection of the prostate (TURP) revealed TZ cancer in 82% and PZ cancer either alone or with TZ cancer in 22%. The 9 patients with PZ cancer in the TURP specimen included 5 of the 11 with extracapsular extension and all 5 of those with seminal vesicle invasion. Every patient with stage B disease had PZ cancer which, in all except 3 cases, was of significantly larger volume and higher grade than any TZ cancer (present in 43%) in the same gland. In patients with stage B cancer, total tumour volume was 5.13 ml with 91% of PZ origin. TZ cancer tended to be well differentiated in all patients, even at large volumes, whereas PZ cancer was often moderately or poorly differentiated even at low volumes. In patients with stage B disease, TZ cancer appeared to be incidental and of no clinical importance, while in stage A patients PZ cancers were sometimes large, poorly differentiated and extended outside the prostate. Progression of a stage A cancer seems more likely to result from PZ cancer than TZ cancer, and the finding of PZ cancer in a TURP specimen should probably preclude its classification as stage A1.

Correlation of ultrasound guided and digitally directed transrectal biopsies of palpable prostatic abnormalities.

The authors evaluated 51 patients with palpable prostatic abnormalities detected during digital rectal examination. These findings consisted of a nodule or an area of induration. Each palpable abnormality was confined to 1 prostatic lobe and there was no suggestion of extracapsular extension of neoplasm or systemic metastatic disease. All patients underwent 7.0 MHz. sagittal ultrasound guided transrectal biopsy followed by digitally directed transrectal biopsy. Biopsies were obtained only from the area of interest. The procedure was performed in the outpatient clinic without use of sedation or anesthesia. Digitally directed biopsies were positive for adenocarcinoma in 9 lesions. Ultrasound guided biopsies detected adenocarcinoma in 23 lesions, including all those detected by the blind digitally directed technique. This study demonstrates greater diagnostic accuracy using 7.0 MHz. ultrasound guided techniques and its routine use is warranted in the evaluation of palpable prostatic abnormalities.

Excretion of high concentrations of cimetidine and ranitidine into rat milk and their effects on milk composition and mammary gland nucleic acid content.

The excretion of cimetidine and ranitidine into rat milk following single or multiple oral doses and the subsequent effects on their suckling pups and on milk composition and milk synthesis were investigated. Following a single dose of [3H]cimetidine, peak milk cimetidine concentrations were maintained from 1 until 4 hr, while plasma concentrations peaked at 10% of the milk level at 30 min and then declined. Multiple doses of cimetidine (18 or 180 mg/kg/day) on Days 13-16 of lactation led to milk cimetidine concentrations of 17 and 113 micrograms/ml. The milk/plasma ratios far exceeded the theoretical milk/plasma ratio of 2.0. Ranitidine concentrations in rat milk following ranitidine treatment (4.5 or 45 mg/kg/day) were also greater (6.8-15 times) than in plasma, but only slightly greater than the predicted ratio of 5.0. There were no changes in liver weight or in hepatic aminopyrine N-demethylase activity in the cimetidine- or ranitidine-treated dams or their pups. Cimetidine treatment had no effect on milk lipid, solid, or protein content, but at 180 mg/kg/day, caused a significant increase in milk lactose. The RNA/DNA ratio in the mammary gland was significantly increased by cimetidine, suggesting increased milk synthesis. Ranitidine had no effect on milk composition or on mammary gland RNA, DNA, or RNA/DNA. Therefore, high concentrations of cimetidine and ranitidine were excreted into rat milk, but no deleterious effects on the suckling pups, or the composition of the milk, or on the milk synthetic activity were observed.

Chromosome 10qter deletion syndrome: a review and report of three new cases.

We report on three patients with terminal deletions of chromosome 10q and compare them to 15 previously reported patients. A similar facial appearance with a prominent beaked nose, large and/or malformed ears, and a pattern of major abnormalities including severe mental retardation, cardiac anomalies, and anogenital anomalies are reviewed. We feel the manifestations of del 10qter are sufficiently distinct to suggest this diagnosis on clinical examination.

Transfer of di(2-ethylhexyl) phthalate through rat milk and effects on milk composition and the mammary gland.

Five daily oral doses of di(2-ethylhexyl) phthalate (DEHP) (2 g/kg) given to rats on Days 2-6, 6-10, or 14-18 of lactation caused significant decreases in body weight and increases in hepatic peroxisomal enzymes palmitoyl CoA oxidase and carnitine acetyltransferase in the dams and their suckling pups. Plasma cholesterol and triglyceride levels were decreased in the lactating dams. Decreased food consumption, as indicated by pair-fed rats, accounted for the decreased body weight in the pups but not the increases in enzyme activities. To determine whether DEHP and mono(2-ethylhexyl) phthalate (MEHP) were transferred through the milk, milk and plasma were collected from lactating rats 6 hr after the third dose of DEHP. The milk contained 216 +/- 23 micrograms/ml DEHP and 25 +/- 6 micrograms/ml MEHP (mean +/- SE), while the plasma contained less than 0.5 micrograms/ml DEHP and 75 +/- 12 micrograms/ml MEHP. The high milk/plasma ratio for DEHP (greater than 200) indicates efficient extraction of DEHP from the plasma into the milk. DEHP dosing during lactation also caused a decrease in mammary gland weight and a decrease in mammary gland RNA content which reflects synthetic activity. The water content of the milk was reduced, which probably accounted for the increase in lipid in the milk. Milk lactose was decreased in DEHP-treated and pair-fed rats, consistent with the decrease in milk production. The results show that exposure to high doses of DEHP during lactation in rats can result in changes in milk quality and quantity and can lead to DEHP and MEHP exposure in the suckling rat pups.

Covalent binding to DNA and mutagenicity of 2,4-diaminotoluene metabolites produced by isolated hepatocytes and 9000 g supernatant from Fischer 344 rats.

Primary cultures of hepatocytes from adult male F344 rats were used to investigate the activation of the hepatocarcinogen 2,4-diaminotoluene (2,4-DAT) to metabolites which bound covalently to DNA. Covalent binding of 2,4-DAT to DNA was significantly greater than that of a non-carcinogenic isomer, 2,6-DAT. Treatment of male rats with 5,6-benzoflavone (BNF), an inducer of cytochrome P-450c and P-450d, had no effect on the binding of 2,4-DAT to DNA of hepatocytes from these animals. However, treatment of hepatocytes in vitro with metyrapone or piperonyl butoxide, two general inhibitors of P-450 enzymes, inhibited the binding of 2,4-DAT to DNA by approximately 80-85%. Two inhibitors of sulfation, pentachlorophenol and 2,5-dichloro-4-nitrophenol, also inhibited DNA binding in hepatocytes from both BNF-induced (91 and 85% respectively) and control rats (82 and 41% respectively), indicating that sulfation may also be required. 2,4-DAT was a more potent mutagen than 2,5- or 2,6-DAT in the Ames Salmonella mutagenesis assay using hepatic S9 fractions from F344 rats as an activating system. In contrast to DNA binding, activation of 2,4-DAT to mutagens by S9 fractions from BNF-treated rats was greater than that by S9 from control rats. The present study shows that 2,4-DAT is activated by hepatocytes of F344 rats to products which bind covalently to DNA. Both cytochrome P-450 and sulfation appear to be involved in the activation.