Enhancing cervical cancer detection using nucleic acid hybridization and acetic acid tests.

Acetowhitening of abnormal cervical epithelium has been suggested as an indicator of increased cervical cancer risk. The presence of human papillomavirus (HPV) types 16, 18, 31, 33 and 35 may also indicate increased cervical cancer risk. Hence, tests that detect these two abnormal conditions may augment that Papanicolaou smear (Pap test) as predictors of cervical cancer risk. The cohort consisted of 145 women aged 14 to 47 (mean 21 years) attending health clinics. Thirty women (20.6 percent) showed acetowhitening of the cervical epithelium following exposure to vinegar of 4-percent acetic acid content. Fourteen (9.6 percent) had a positive Pap test and 13 (9 percent) carried a cervical HPV infection as determined by the commercially available ViraPap and ViraType nucleic acid tests. Statistical analysis of the data showed a positive correlation between Pap, ViraPap and acetic acid tests results. The acetic acid test and the nucleic acid tests were the sole positive tests for 21 (14.5 percent) and nine (6.2 percent) women, respectively. Four women with negative Pap results were infected with HPV types previously shown to have an association with cervical intraepithelial neoplasias, carcinoma in situ and cervical cancer. The authors have concluded that the acetic acid and nucleic acid tests detect women at risk for cervical cancer who would not have been detected by the Pap test alone.

Effect of 1,3-dinitrobenzene on prepubertal, pubertal, and adult mouse spermatogenesis.

Exposure of prepubertal, pubertal, and adult mice to 0, 8, 16, 32, 40, or 48 mg 1,3-dinitrobenzene (m-DNB)/kg body weight and measuring responses 1-25 d posttreatment (dpt) demonstrated significant effects on testicular function only at 48 mg/kg dosage. m-DNB had no effect on body or testis weights with the exception of reduced adult mouse testis weights at 22 dpt with 48 mg/kg (p less than .05). None of the exposures resulted in detectable levels of germinal epithelial cells in the ductus epididymis. Exposure of prepubertal and pubertal mice to m-DNB caused only minimal nonsignificant changes in the relative percent of testicular cell types present up to 25 dpt. The adult mice testicular cell type ratios, in particular the round and elongating spermatid populations, changed significantly at doses of 48 mg/kg. Also, a reduction in the percent tetraploid cells occurred at d 1, suggesting these cells may be a primary target of m-DNB action. Caput and caudal sperm from mice exposed to m-DNB prior to puberty did not demonstrate an increased susceptibility to DNA denaturation when analyzed by the sperm chromatin structure assay. However, in pubertal mice, m-DNB exposure further exaggerated the abnormal chromatin structure that normally characterizes sperm during the onset of sperm production. In adult mice, 48 mg/kg resulted in increased susceptibility to DNA denaturation of caput sperm chromatin at 11 dpt (p less than .05) and in caudal sperm at 22 dpt (p less than .01). The abnormal chromatin structure of cauda sperm from adult mice was highly correlated with sperm head morphology abnormalities (ABN; 0.82 to 0.95, p less than .01, 11 and 22 dpt, respectively), but showed lower correlations with dose (0.60 to 0.79, p less than .01, 11 and 22 dpt, respectively). For pubertal mice, a positive relationship was also observed between the variation of sperm chromatin structure abnormalities and ABN. The effect of m-DNB on testicular function in prepubertal and pubertal mice appear to be less pronounced than in adult mice. Furthermore, following exposure to the same dosage, the effect of m-DNB is less severe in adult mice than that observed for adult rats as reported in the companion paper.

Flow cytometric analysis of effects of 1,3-dinitrobenzene on rat spermatogenesis.

Exposure of 100-d old rats to 1,3-dinitrobenzene (m-DNB) at dosages up to 48 mg/kg resulted in disruption of spermatogenesis as measured by flow cytometry (FCM) of acridine orange-stained sperm and testis cells. One day (d 1) after a single exposure to 48 mg/kg m-DNB, FCM measurements of caput epididymal fluid cells demonstrated the presence of testicular germinal epithelial cells apparently sloughed off into the epididymis. Also, at d 1 after the same exposure, a decrease in pachytene spermatocytes was observed. By d 16 after exposure to 32 or 48 mg/kg, testicular damage was evidenced by an alteration of cell type ratios in FCM-analyzed populations of testicular cells. Extensive recovery of cell type ratios occurred by d 32. At d 16, dosages of 32 and 48 mg/kg caused alterations of sperm chromatin structure as determined by the flow cytometric sperm chromatin structure assay (SCSA); 48 mg/kg caused alterations at both d 16 and d 32. Exposure to m-DNB caused a dose response increase in percent sperm head morphology abnormalities (%ABN) assessed in cauda epididymal and vas sperm. A slightly higher correlation existed between dose and SCSA alpha t values (d 16, .78; p less than .01) than between dose and %ABN (d 16, .70; p less than .01). Also, a higher correlation existed between standard deviation of alpha t (SD alpha t) values and %ABN (.97; p less than .01) than between dose and %ABN (.70; p less than .01). This study demonstrated rapid and unique FCM procedures originally derived for reproductive toxicology studies in mice to be equally useful for studies in rats.

Effects of the fungicide methyl-benzimidazol-2-yl carbamate (MBC) on mouse germ cells as determined by flow cytometry.

Dual-parameter (DNA, RNA) flow cytometry (FCM) measurements were made on testicular and epididymal sperm cells isolated from mice exposed by oral gavage to 0, 250, 500, or 1000 mg/kg X 5 d of the fungicide methylbenzimidazol-2-yl carbamate (MBC), which is known to bind with tubulin subunits and inhibit polymerization and microtubule formation. Effects of exposure to MBC were measured at 7, 24, and 39 d posttreatment. MBC had no effect on body weights, but testis weights and sperm parameters were altered, with few exceptions, only at the highest exposure level. Testis weights were reduced by about 25% at 7 and 24 d after exposure; recovery was observed by 39 d after treatment. FCM measurements of testicular cells showed relative percentages of certain testicular populations (round, elongating, and elongated spermatids) were different from the control pattern 7 and 24 d after treatment. The mean percent of cauda epididymal sperm head morphology abnormalities and the susceptibility of the nuclear DNA to denaturation were both elevated at 7, 24, and 39 d after exposure to 1000 mg/kg. The level of denaturation was determined by FCM measurements of the metachromatic shift in acridine orange (AO) stained sperm nuclei from green (native DNA) to red (single-stranded DNA) fluorescence and quantitated by the expression alpha t[red/(red + green] fluorescence. These data demonstrate that spermatogenesis is sensitive to high-dose MBC exposure resulting in an altered ratio of testicular cell types present, abnormal sperm head morphology, and an altered sperm chromatin structure.

Mouse testicular and sperm cell development characterized from birth to adulthood by dual parameter flow cytometry.

Dual parameter flow cytometry was used to investigate cellular changes in male germinal tissue during normal postpartum maturation in B6C3F1/J mice. Animals were killed at 2-day intervals from 2 to 42 days postpartum and at 48, 64, 72, 93 and 100 days postpartum. Testicular, cauda epididymis and vas deferens cell suspensions were stained with the metachromatic fluorochrome acridine orange and measured by flow cytometry for red and green fluorescence levels after excitation by blue laser light. Intensities of red and green fluorescence reflect amounts of single- and double-strand nucleic acid sites available for acridine orange staining, respectively, and were used to classify cells on the basis of ploidy level, RNA content, and chromatin structure, as defined by susceptibility to acid denaturation of DNA in situ. Sperm from cauda epididymis and vas deferens were examined by light microscopy to determine frequency of abnormal sperm head morphology. Fluorescence data derived from acridine orange-stained testicular cells quantified the sequential changes in 1) proportions of haploid, diploid and tetraploid cell types during the first round of spermatogenesis, and 2) proportions of round, elongating, and elongated spermatids during the first round of spermiogenesis. Ratios of the three major testicular populations (haploid, diploid, and tetraploid) reached adult levels by 48 days postpartum. Sperm cells were first detected in the cauda epididymis and vas deferens on 30 and 36 days postpartum, respectively. Early sperm populations, compared to adult sperm, exhibited up to 89% abnormalities in sperm head morphology that correlated with significant levels of abnormal chromatin structure. Percentage of sperm head abnormalities and chromatin structure in the cauda epididymis and vas deferens approached normal adult levels by 42 and 48 days postpartum, respectively.

Heterogeneity of lethals in a "simple" lethal complementation group.

Of 24 ethyl methanesulphonate-induced, recessive-lethal mutations in the region 9E1-9F13 of the X chromosome of Drosophila melanogaster, eight fall into a typically homogeneous lethal complementation group associated with the raspberry (ras) locus. Mutations in this group have previously been shown to be pleiotropic, affecting not only ras but also two other genetic entities, gua 1 and pur 1, which yield auxotrophic mutations.--The eight new mutations have been characterized phenotypically in double heterozygotes with gua 1, pur 1 and ras mutations. Despite their homogeneity in lethal complementation tests, the mutations prove quite diverse. For example, two mutations have little or no effect on eye color in double heterozygotes with ras2. The differences between the lethals are allele-specific and cannot be explained as a trivial outcome of a hypomorphic series.--Taken alone, the lethal complementation studies mask the complexity of the locus and the diversity of its recessive lethal alleles. By extension, we argue that the general use of lethal saturation studies provides an unduly simplified image of genetic organization. We suggest that the reason why recessive lethal mutations rarely present complex complementation patterns is that complex loci tend to produce mutations that affect several subfunctions.

Hypomorphic lethal mutations and their implications for the interpretation of lethal complementation studies in Drosophila.

In a small region of the X chromosome of Drosophila melanogaster, we have found that a third of the mutations that appear to act as lethals in segmental haploids are viable in homozygous mutant individuals. These viable mutations fall into four complementation groups. The most reasonable explanation of these mutations is that they are a subset of functionally hypomorphic alleles of essential genes: hypomorphic mutations with activity levels above a threshold required for survival, but below twice that level, should behave in this manner. We refer to these mutations as "haplo-specific lethal mutations." In studies of autosomal lethals, haplo-specific lethal mutations can be included in lethal complementation tests without being identified as such. Accidental inclusion of disguised haplo-specific lethals in autosomal complementation tests will generate spurious examples of interallelic complementation.