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Sister chromatid exchange (SCE) techniques were used to analyze the genetic effects of ethyl carbamate (urethane) in cultured mouse-bone marrow cells, and in several different mouse tissues in vivo. Ethyl carbamate concentrations up to 5.0 mg/ml were ineffective in causing a significant elevation of SCE in vitro. After in vivo drug administration, bone marrow, liver and spermatogonial cells all revealed significant dose-related increases in SCE. Baseline and relative incremental levels of SCE were somatic vs germ tissue-specific. Regenerating liver cells exhibited significantly greater absolute SCE values than all other tissues examined. Marrow cells revealed intermediate values, while germ cells were the least sensitive in SCE responsiveness. Spermatogonia required a fourfold higher dose, over that effective in somatic tissues, to promote an approximate doubling of the baseline SCE level. In vivo SCE analysis affords sensitive risk assessments for different tissues. Thus, this approach; should be generally useful for studying compounds with questionable mutagenic potential, and/or those exerting target organ specificities of related biological activity (eg, toxicity, carcinogenesis).

The effectiveness of benzo(a)pyrene [B(a)P]-DNA binding as an internal dosimeter was evaluated. Data were obtained from concurrent studies, measuring B(a)P induced genotoxic effects and DNA adducts in several short-term bioassay systems: cytotoxicity, gene mutation, and sister chromatid exchange in Chinese hamster V79 cells; cytotoxicity, gene mutation, and chromosome aberrations in mouse lymphoma L5178Y TK+/-; cytotoxicity and enhanced virus transformation in Syrian hamster embryo cells; and cytotoxicity and morphological transformation in C3H10T1/2CL8 mouse embryo fibroblasts. Both total B(a)P-DNA binding and specific B(a)P-DNA adducts were measured. N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl)deoxyguanosine [BPDE I-dGuo] was one of the major adducts identified in all bioassay systems. DNA binding and genotoxic responses varied significantly between bioassays. Each genetic end point was induced with a differing efficiency on a per adduct basis. However, the relationships between frequency of genetic effect or morphological transformation and B(a)P-DNA binding or BPDE I-dGuo were linear within a given assay. In order to compare biological end points of diverse frequencies in diverse biological systems, a doubling adduct level, expressed as the number of BPDE I-dGuo adducts per unit of DNA required to double the induced frequency of biological response, was applied to the data.

Experiments were designed to investigate the induction of micronuclei (MN) in mouse peripheral blood lymphocytes (PBLs) after in vitro or in vivo exposure to 60Co gamma radiation. For the in vitro experiments, 4 ml of blood from male C57BL/6J mice were either irradiated in 6 ml Falcon culture tubes as whole blood or isolated to obtain mononuclear leukocytes (MNLs) that were pelleted by centrifugation and then irradiated in RPMI 1640. For the in vivo analysis, mice received whole body irradiation, blood was obtained by cardiac puncture, and the MNLs were isolated for each mouse. Exposures were at a rate of 0.82 to 0.90 Gy/min to yield doses of 0.5, 1, 2, 3, or 4 Gy. MNLs were cultured using cytochalasin B for MN analysis in binucleated PBLs. There was a significant dose-dependent increase in MN observed at all doses. Dose-response curves for the in vivo and in vitro whole blood experiment were not significantly different. However, for isolated pelleted MNLs irradiated in vitro, the MN frequency at 4 Gy was less than one-half that seen in the in vivo experiment. The large difference in MN response is thought to be due to the radioprotective effect of hypoxia.

In December 1972, an 18-year-old male apparently took an accidental overdose of heroin. After becoming ill, he attempted to receive treatment for his condition by being admitted to the emergency room of Wahiawa General Hospital. When confronted by attending physicians in the emergency room as to the nature of his illness, the youth stated that he had only eaten 10 hallucinogenic mushrooms (an average dose for Copelandia cyanescens, the mushroom in question) and nothing else. This story was most likely concocted either by the youth himself or by some of his friends, who probably thought that they could or would be prosecuted for the use of an illegal substance. It is not uncommon that when confronted by an overdose of drugs, many young people are afraid to report their illness to the proper medical authorities out of fear of prosecution due to their illegal activities (Young et al. 1982). The doctors who attempted to treat this young man should not be held liable for his death, even though their treatment of the patient was more supportive (i.e., the talk-down method) than pharmacological. They had no way of knowing that the patient had lied to them about his condition. While it is true that several doctors had diagnosed Gomilla as possibly suffering from muscarine poisoning, why was no atropine or scopolamine administered to the patient? And why was his stomach not pumped and specimens collected for a toxicologist to study for the presence of toxins?(ABSTRACT TRUNCATED AT 250 WORDS)

The industrial chemical acrylamide is suspected to induce potentially heritable genetic damage. While several studies in rodents have indicated that this substance can damage spermiogenic cells, resulting in dominant lethals and heritable translocations, cytogenetic assessments of premeiotic and meiotic cells after exposure have produced equivocal results. In the present study, various cytogenetic endpoints in both somatic and germ-line cells from acrylamide-treated mice were evaluated. Sister chromatid exchanges and micronuclei, but not chromosome aberrations, were induced in spleen cells; synaptonemal complex irregularities (asynapsis), but not chromosome aberrations, were induced in germ cells.

A small tablet of 5-bromodeoxyuridine (BrdU), implanted subcutaneously in a mouse, provides sustained release of base analog sufficient to effect substitution of DNA throughout an entire replication period. As illustrated by studies of mouse bone-marrow and spleen cells in the presence or absence of cyclophosphamide, this depot method of BrdU administration greatly simplifies in vivo analysis of sister-chromatid-exchange formation.

Seven patients with aortic regurgitation, manifesting diastolic flutter of the aortic valve cusps (DFAVC) in the echograms, are described. Five patients with infective endocarditis revealed coarse or fine, irregular DFAVC. Two patients with severe aortic regurgitation and a musical murmur manifested regular DFAVC with a frequency identical to that of a simultaneously recorded diastolic murmur. Of the 5 patients with infective endocarditis, 4 required urgent aortic valve replacement and 1 died. The 2 patients with musical murmurs are clinically stable without surgery. This report extends the clinical spectrum of patients with DFAVC and describes the character of the flutter in patients with muscial murmurs. Furthermore, it suggests that DFAVC is a sign of severe aortic regurgitation.

Echocardiography is an established method for the diagnosis of pericardial effusion. Echographic findings in fibroadhesive disease have also been described. The interpretation of a changing serial echogram during the clinical course of pericardial disease has not been established. Sixteen patients with echographic evidence of pericardial effusion were followed with serial echographic studies. In seven, surgical or autopsy correlation was obtained. Because of the serial changes noted we undertook a study in dogs to clarify the problem of variability in intensity of sound reflected from the pericardial space. Three open-chested dogs were studied with rapid sequence surface echograms as blood introduced into the pericardial space was converted from the unclotted to the clotted state. In all three dogs blood clooting increased the intensity or sound reflected from the pericardial space. Our study of two patients with fibroadhesive pericardial disease documents serial changes in echoes from the pericardium and pericardial space accompanying the clinical evolution of the disease process, and suggests a method for avoiding the commonly encountered difficulty in proper identification of the pericardial and epicardial echoes. Our preliminary studies suggest that serial echograms should play an important role in the management of patients with pericardial effusion; further surgical/pathological correlations are required.

Studies were conducted in 189 oral surgery out-patients to evaluate the combination of meperidine hydrochloride and diazepam for anesthesia and to determine its effect on the cardiovascular and pulmonary systems. Results showed that neither ventricular cardiac arrhythmias nor clinically significant respiratory depression occurred in the sedated patients; meperidine-diazepam proved to be satisfactory for oral surgical procedures.

Methyl isocyanate (MIC) was tested for genetic toxicity in a variety of in vitro and in vivo assays. Negative results were obtained in the Salmonella/mammalian microsome assay using five bacterial strains in a preincubation protocol. The Drosophila sex-linked recessive lethal test also gave negative results in studies that involved three routes of administration: inhalation, feeding, and injection. Positive results were obtained for three endpoints in cultured mammalian cells. Reproducible, dose-related increases in trifluorothymidine-resistant clones were induced in L5178Y mouse lymphoma cells, and the frequencies of both SCE and chromosomal aberrations increased in Chinese hamster ovary cells. These effects were independent of exogenous metabolism. In mice exposed to methyl isocyanate by inhalation, cytogenetic analyses were carried out on bone marrow, blood, and lung cells. A single, 2-hr exposure to concentrations of 0, 3, 10, and 30 ppm MIC produced no evidence of chromosomal effects in the bone marrow, although significant cell cycle delay was observed. In four experiments involving exposures on 4 consecutive days to 0, 1, 3, or 6 ppm, delays in bone marrow cell cycle were again observed. Increases in SCE and chromosomal aberrations were observed in bone marrow cells, and a dose-related increase in SCE occurred in lung cells but not in peripheral blood lymphocytes. A significant increase in micronucleated polychromatic erythrocytes in the peripheral blood was observed in male mice in one experiment. From these results, it appears that methyl isocyanate has the capacity to affect chromosome structure but not to induce gene mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

As heritable chromosome anomalies are implicated in a variety of human disabilities, their induction in germ cells by environmental chemicals is viewed as a threat to health (National Research Council, 1982; Hook, 1983). Synaptonemal complex (SC) analysis is a novel approach for the detection of germ-line chromosomal damage. This sensitive cytological procedure reveals induced structural damage and pairing abnormalities in SCs of meiotic prophase chromosomes, together with other germ-line toxic effects, in the testes of rodents treated with mitomycin C and cyclophosphamide. Our results demonstrate the effectiveness of SC analysis as a rapid and practical in vivo germ-line mutagen assay that lacks many of the short-comings of existing tests.

Meiotic cells have been, and remain, a relatively difficult tissue in which to study SCEs. Experimental evaluations have had to contend with various problems unique to germ cells, i.e., poor in vitro growth, unusual cytotoxicities (from BrdUrd), trying chromosome morphologies, and confusion between chromosome label patterns generated by SCEs, COs, and replication kinetics. Nevertheless, BrdUrd differential staining techniques in insects and rodents have progressed to the point where SCE frequencies can be reliably determined. In the described Armenian hamster system, SCEs may be directly resolved in selected primary spermatocyte bivalents and indirectly assessed in secondary spermatocyte cells. SCE frequencies determined from spermatogonial, primary spermatocyte, and secondary spermatocyte cells are in good agreement. The system should be applicable for studies of possible mechanistic similarities between SCE and CO exchange, and for evaluating genotoxic effects. Experimental evidence in Drosophila (33) argues against a very close molecular relationship between these forms of breakage and recombination. However, it is likely that new information can be gained from cells in which normal SCE and CO frequencies and distributions can be characterized, and from studies of the consequences to these events after various mutagenic perturbations to premeiotic and meiotic DNA.

5-Bromodeoxyuridine (BrdU) tablets with different physical characteristics are useful in a wide variety of studies requiring detection of DNA replication in vivo. These tablets can effect a high substitution of BrdU in DNA, thereby permitting sister chromatid differentiation in chromosomes stained with 33258 Hoechst alone or in conjunction with Giemsa. Baseline and cyclophosphamide-induced in vivo sister chromatid exchange frequencies in mouse spleen, marrow, and thymus were measured and found to be significantly greater than those in spermatogonia. Sister chromatid exchange analysis was also extended to mouse liver and to Chinese hamster and Armenian hamster marrow cells. Sister chromatid differentiation was observed in Armenian hamster meiotic tissue, and evidence for interhomolog chromatid exchange obtained.

Male Armenian hamsters were evaluated for age effects upon meiotic recombination and aneuploidy incidence. Primary spermatocytes from young and old animals revealed similar chiasma frequencies. The incidence of terminal-type chiasmata in sex bivalents was also measured and found to be unaffected by age. Percentages of univalents and of hyperploid metaphase II cells were evaluated by chromosome groups based upon individual chromosome morphologies. Regardless of age, the larger dichiasmate chromosomes were seldom observed as univalents, or as extra chromosomes in hyperploid cells. The monochiasmate small autosomes and sex chromosomes revealed univalent frequencies approximating to 5.5-6.5% in animals from both age groups. However, only the small autosomes appeared to be significantly implicated in hyperploidy. Although still a rare event, metaphase II hyperploidy was significantly increased in older animals (0.9% incidence in young and 2.4% in old hamsters). The events leading to higher aneuploidy levels in older male Armenian hamsters are not clear. A role for reduced meiotic recombination as a function of age cannot be ruled out in the present study. However, there was no support for this hypothesis, and consideration of other possible mechanisms of age effects to increase aneuploidy levels is indicated.

In vivo BrdU-dye methodology is used to reveal patterns of replication kinetics and meiotic recombination in male germ cells of the Armenian hamster. Analogue substitution over a fraction of the DNA synthesis period results in distinctive 33258 Hoechst fluorescence staining of late replicating chromatin, detectable at spermatogonial or spermatocyte I and II stages. Spermatogonial cells which are extensively substituted with BrdU over the penultimate synthesis period reveal sister chromatid differentiation in all chromosomes of primary or secondary spermatocytes. In chiasmatic regions, exchanges between unlike-stained non-sister chromatids are indicated by isolatelled segments, while those occurring between like-stained non-sister chromatids are not directly detected. In sex chromosomes from young animals, these alternative images occur in a ratio of 1:1, which supports the concept that homologue non-sister chromatid regions are randomly broken and re-united in the process of chiasma formation. Deviations from randomness appeared to occur in older animals. Sex bivalent chiasmata are either coincident with points of visible exchange, or they appear to have variable degrees of terminalization. Secondary spermatocytes display sharp chromatid contrast, and aid in mapping the positions and frequencies of homologue exchanges.

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 micron with a peak value around 0.3 to 0.6 micron regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments.(ABSTRACT TRUNCATED AT 250 WORDS)