Mark E. Hurtt

Degeneration and regeneration of the olfactory epithelium following inhalation exposure to methyl bromide: Pathology, cell kinetics, and olfactory function☆

The effects of acute inhalation exposure to methyl bromide (MeBr) on the olfactory epithelium of male F-344 rats was investigated by morphologic examination of animals killed at varying timepoints during and following exposure to 200 ppm MeBr 6 hr/day for 5 days. Cell replication rate and histopathology were used to assess the kinetics of repair. In addition, olfactory function, using the buried food pellet test, was assessed and the result compared with morphological recovery. Extensive destruction of the olfactory epithelium was evident in animals killed directly after a single 6-hr exposure to MeBr. Histologic features of these lesions indicate that the primary, or most severe, effect of MeBr exposure was on the sustentacular cells and mature sensory cells; basal cells were generally unaffected. By Day 3, despite continued exposure, there was replacement of the olfactory epithelium by a squamous cell layer that increased in thickness and basophilic cytoplasmic staining over the next 2 days of exposure. One week postexposure, the epithelial region was covered by a layer of polyhedral, basophilic cells, and from 2 to 10 weeks postexposure, the epithelium exhibited progressive reorganization to reform the original olfactory epithelium pattern. By Week 10, 75-80% of the olfactory epithelium appeared morphologically normal. Cell replication showed a single peak of olfactory epithelial cell proliferation at Day 3 of exposure, with a labeling index of 14.5% compared to 0.7% in controls. Cell replication rates returned gradually to control levels by Week 10 postexposure. Behavioral tests of olfactory function in animals after a single 6-hr exposure to 200 ppm MeBr demonstrated a loss of the sense of smell, with recovery of this function by Day 6. Exposure to 90 ppm caused no observable effect on olfactory function or morphology. These findings demonstrate that the olfactory mucosa is highly sensitive to the toxic effects of MeBr and that olfactory epithelial cell proliferation, and possible regeneration, begins and occurs rapidly even in the face of continued exposure. Cell replication was most prominent in the layer of basal cells adjacent to the basal lamina, supporting proposals by other workers that the progenitors of both sustentacular cells and neurons reside in this location. Of interest is the fact that functional recovery occurs prior to complete morphological reorganization, indicating the shortcoming of utilizing olfactory morphology as an index of functional integrity.

Differences in the quality of semen in outdoor workers during summer and winter.

Abstract Background and Methods. In warm climates throughout the world, there is a deficit of births during the spring season. To determine whether this deficit might reflect a deleterious effect of heat on the male reproductive capacity during the previous summer, we obtained semen specimens in summer and winter from normal men who worked outdoors in the vicinity of San Antonio, Texas, and we performed automated semen analyses with an image-analysis system. Results. Pairwise comparisons among 131 men without azoospermia who contributed specimens in both summer and winter revealed significant reductions during the summer in sperm concentration, total sperm count per ejaculate, and concentration of motile sperm. The mean decreases in these values after adjustment for potential confounding characteristics were 32 percent (95 percent confidence limits, 28 and 44 percent), 24 percent (95 percent confidence limits, 18 and 43 percent), and 28 percent (95 percent confidence limits, 24 and 44 percent), respective...

Unit length as the denominator for quantitation of cell proliferation in nasal epithelia.

Cell proliferation data, generally based on a labeling index (LI), provide a valuable endpoint for assessment of toxic and potentially carcinogenic responses in laboratory animals. Measurement of the LI is time consuming because of the large number of cells that need to be counted to determine the denominator. In respiratory mucosa, the total cell count of the surface epithelia may be altered in response to treatment, either through cell loss or increases in cell number (e.g., hyperplasia). As an alternative to the more conventional LI, the present studies were carried out to assess the value of expressing cell proliferation in nasal epithelia as a unit length labeling index (ULLI), defined as labeled cells per mm of basement membrane. Rats were exposed by inhalation to formaldehyde or methyl bromide, and changes in cell proliferation were determined in the respiratory and olfactory epithelia, respectively, using both total cell count and basement membrane length as denominators. Total cell counts were clearly influenced by treatment, while basement membrane length was not. Both methods revealed similar treatment-induced effects on cell proliferation, and in fact were highly correlated (R ≥ 0.92, p < 0.001). It was concluded that the ULLI method provides an effective alternative to total cell counts and the LI method. This approach is not influenced by alterations in the total cell population, and has the benefit of being less labor intensive than LI determinations.

Histopathology of Acute Toxic Responses in Selected Tissues from Rats Exposed by Inhalation to Methyl Bromide

To determine and characterize the histological changes induced in selected tissues from the Fischer 344 rat by acute inhalation exposure to methyl bromide (MeBr), groups of 10 male rats (11-13 weeks old) were exposed to 0, 90, 175, 250, or 325 ppm MeBr 6 hr/day for 5 days. Animals were anesthetized with phenobarbital then perfusion-fixed 1-2 hr after the last exposure or in extremis (325 ppm, 4 days) with Karnovskys fixative and selected tissues were processed for light microscopy. With the exception of the nasal passages, tissues were selected on the basis of previous studies with methyl chloride (MeCl). The nose was examined as part of ongoing research of nasal toxicity in this laboratory. The principal clinical signs, confined to the 250 and 325 ppm groups, were diarrhea, hemoglobinuria, and, in a few cases, gait disturbances and convulsions. A dose-dependent vacuolar degeneration of the zona fasciculata of the adrenal glands, cerebellar granule cell degeneration, and nasal olfactory sensory cell degeneration were seen in all concentration groups except at 90 ppm. Cerebral cortical degeneration and minor alterations in testicular histology were seen only in the 325 ppm group. Hepatocellular degeneration was confined to the 250 and 325 ppm groups. No changes were seen in the kidneys or epididymides. This study demonstrates that MeBr has similar target organs to MeCl suggesting that similar mechanisms of toxicity may be operational. However, important differences in the nature of cellular responses to these chemicals may facilitate studies on their mechanisms of actions.

Computer-assisted semen analysis: results vary across technicians who prepare videotapes

Automated semen analyses revealed differences of 21% to 30% in concentration-related parameters and 5% to 11% in motion-related parameters between means of groups of replicate specimens. Disparities among videotapes produced by two laboratory technicians accounted for the divergence in concentration-related parameters. This resulted partially from differences between the two technicians in propensity to dilute concentrated specimens. The causes of the greater portion of disparities between videotaping technicians, however, have not been identified. Differences in motion-related parameters could not be ascribed to technicians, but the basis for these differences is also unknown. The results suggest that values obtained from the CellSoft image analysis system may not be comparable between technicians or laboratories, despite use of identical computer parameter settings. Until effective quality control measures have been implemented, such comparisons must be made with caution.

Role of testicular versus epididymal toxicity in the induction of cytotoxic damage in fischer-344 rat sperm by methyl chloride

Exposure of male Fischer-344 (F-344) rats to methyl chloride (MeCl) results in testicular and epididymal toxicity and the induction of both pre- and postimplantation embryonic loss; the preimplantation loss is caused by cytotoxic damage to sperm that leads to failure of fertilization (Toxicol Appl Pharmacol 1986; 86:124-130). The present study examined whether the cytotoxicity of MeCl to sperm is due to the testicular or epididymal toxicity of MeCl. Groups of 18 males were exposed to 3000 ppm MeCl 6 h/day for 5 days, with and without concurrent treatment with the anti-inflammatory agent 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW755C; 10 mg/kg, i.p. 1 h pre- and postexposure); BW755C was used to inhibit the epididymal toxicity of MeCl. Control groups were untreated or injected as described above with BW755C. Six males from each group were killed weekly for 3 weeks. Toxic effects of MeCl on the testis were demonstrated by decreased relative organ weight (week 3), testicular histopathology (weeks 1-3) and decreased daily sperm production (weeks 1-3); these effects were not prevented by BW755C. In both the MeCl and the MeCl + BW755C treatment groups, tubules devoid of sperm were observed in regions 4 and 5 of the epididymis at week 2, and in regions 6A and 6B at week 3. Sperm were present in the vas deferens of both groups at week 3 in decreased numbers and had decreased motility and more frequent morphologic abnormalities compared to untreated controls. In conjunction with known epididymal transit times for F-344 rat sperm, these data indicate that the induction of preimplantation loss by MeCl at weeks 2 and 3 postexposure is likely to result from cytotoxic effects on sperm located in the testes at the time of exposure.

Evaluation of Spermatogenesis and Sperm Quality in the Rat following Acute Inhalation Exposure to Methyl Bromide

Methyl chloride (MeCl) and methyl bromide (MeBr) have similar target organ specificities in the male F-344 rat, and MeCl is a known reproductive toxicant in that species. Recently, both acute and subchronic inhalation exposures to MeBr were found to produce varying degrees of testicular alteration (S.L. Eustis, S.B. Haber, R.T. Drew, and R.S.H. Yang, 1986, Toxicologist, 6, 54; N. Kato, S. Morinobu, and S. Ishizu, 1986, Ind. Health, 24, 87-103; M.E. Hurtt, K.T. Morgan, and P.K. Working, 1987, Fundam. Appl. Toxicol., 9, 352-365). The present study examined the reproductive effects of MeBr in the male F-344 rat. Adult males (11-13 weeks) were exposed by inhalation to 0 or 200 ppm MeBr 6 hr/day for 5 days (first day of exposure = Day 1). Ten animals from each group were anesthetized with pentobarbital and terminated on Days 1, 3, 5, and 8. Additionally, five males from each group were killed on Days 6, 10, 17, 24, 38, 52, and 73. Plasma testosterone concentration was reduced during and immediately following exposure (Days 1, 3, 5, and 6), but returned to control levels by Day 8. Nonprotein sulfhydryl (NPSH) content of the liver and testis was reduced during exposure but returned to control levels by Day 8 (3 days postexposure). No other reproductive indices, including testis weight, daily sperm production, cauda epididymal sperm count, sperm morphology, percentage motile sperm, linear sperm velocity, and epididymal and testicular histology, were affected by MeBr exposure at any time point examined. Thus, although MeBr causes a transient decrease in plasma testosterone and testicular NPSH concentrations during acute exposure, it has no lasting effect on sperm quality or spermatogenesis in the F-344 rat.