Application of computer-assisted sperm analysis system to elucidate lack of effects of cyclophosphamide on rat epididymal sperm motion.

A computer-assisted sperm analysis (CASA) system was used to examine the motion of epididymal spermatozoa derived from cyclophosphamide (CP)-treated male rats. Male rats were orally dosed daily for 1 week with 20 mg/kg of CP. Males were euthanized or were mated 3 times with untreated females at 1 day, 3 weeks, and 8 weeks after the final treatment. Significant decreases in testicular and epididymal weights and epididymal sperm counts of the treated animals were noted after 8-week recovery. Histopathological morphometry of the testis revealed minimal damage to spermatogonia at 1 day after the final treatment and to spermatocytes after 3-week recovery in the CP-treated group. On Caesarian section, increased post-implantation losses were found in females mated with CP-treated males in matings starting 1 day and 3 weeks after the final treatment. On the other hand, none of the sperm motion parameters of treated males derived from the CASA system exhibited significant changes at any time points, although the spermatozoa of treated males at 1 day and 3 weeks after the final treatment were damaged at the DNA level, and the spermatozoa of males after 8-week recovery had been the target cells of CP when they were spermatogonia in the testis. It was thus found that damaged spermatozoa could exhibit no changes on their motion when the damage was confined to the nuclei, and that the effect of CP on sperm nuclei was reversible.

Dissection and weighing of accessory sex glands after formalin fixation, and a 5-day assay using young mature rats are reliable and feasible in the Hershberger assay.

The rodent Hershberger assay has been used predominantly by the pharmaceutical industry to evaluate androgenic and antiandrogenic chemicals for potential therapeutic use. However, this assay has not yet been formally validated and standardized for use in toxicology testing. There are many variations in the protocol used for this assay. The weight of androgen-dependent tissues is a definitive endpoint in the Hershberger assay. To find out the reliable assay protocol with feasibility, although many possible factors may affect assay reliability, the present study consist of a series of three separate experiments focused on method of dissection and weighing of accessory sex glands (ASGs: ventral and dorso-lateral prostate, seminal vesicles together with coagulating glands, and Cowper's glands), animal age and number of doses. Furthermore, male pubertal assay, an alternative to the Hershberger assay, was also examined its reliability. Experiment 1 explored whether reliably accurate ASG weights can be obtained after formalin fixation. The ASGs were collected from castrated male rats (11 weeks of age) treated daily with corn oil, or testosterone propionate (TP, 0.25 mg/kg/day, s.c.) and p,p'-DDE (0 or 100 mg/kg/day, p.o.) for 5 days. One day after the final treatment, the ASGs were removed carefully and weighed separately, and then fixed overnight in a 10% neutral-buffered formalin and weighed again. After that, the tissues were dried overnight in an oven and weighed again. A high correlation between fresh and fixed tissue weights, and a high correlation between fixed and dried tissue weights were noted. The changes in the tissue weight due to fixation were marginal and were proportional to the fresh weights of the individual tissue. Standard deviation of the fixed tissue weight in each group and the magnitude of responses to TP or p,p'-DDE in fixed tissues were equivalent to those in fresh or dried tissues. These findings indicate that formalin fixation does not interfere with interpretation of assay results, and this procedure was used in the subsequent experiments. Experiments 2 and 3 explored whether animal age or treatment duration altered assay sensitivity. In Experiment 2, antiandrogenic effect of p,p'-DDE (100 mg/kg/day) was detected after 5-and 10-day treatment irrespective of animal age (7 vs 11 weeks). In Experiment 3, antiandrogenic effects of flutamide (1 and 10 mg/kg/day) and p,p'-DDE (10 and 100 mg/kg/day) were compared between two different protocols, the 10-day assay using peripubertal rats and the 5-day assay using young mature rats. Results demonstrated that both protocols could significantly detect antiandrogenic effects of flutamide and p,p'-DDE. These findings demonstrate that (1) dissection and weighing of ASGs after formalin fixation is reliable in the Hershberger assay, (2) when this procedure is used, the 5-day Hershberger assay using young mature rats, expected to be more feasible assay than the 10-day assay using peripubertal rats, is also reliable as well as the 10-day assay using peripubertal rats. Furthermore, we confirmed that male pubertal assay with use of dissection and weighing of fixed tissues also reliable.

Evaluation of a 5-day Hershberger assay using young mature male rats: methyltestosterone and p,p'-DDE, but not fenitrothion, exhibited androgenic or antiandrogenic activity in vivo.

A 5-day Hershberger assay using young mature male rats to detect compounds interfering with androgen receptor (AR)-mediated mechanisms was evaluated for ability to identify p,p'-DDE (a weak AR antagonist) and methyltestosterone (MT, an AR agonist). Fenitrothion, an organophosphate pesticide, was also evaluated in this validated assay. Castrated male Crj:CD(SD)IGS rats (1 week after castration, 11 weeks of age) were subjected to experiments. To determine a suitable value of testosterone propionate (TP) as a reference androgen for detection of antiandrogenic chemicals, castrated male rats were treated daily with TP (0, 0.06, 0.25, 1, 4, or 16 mg/kg/day, s.c.). TP produced increases in weights of ventral prostate, seminal vesicles and levator ani plus bulbocavernosus muscles. Serum androgen level measured by RIA kit (mostly TP) were elevated in a dose-related manner, while the weights of organs with 1 mg/kg/day of TP were nearly equivalent to the maximum responses (i.e., sub-maximal). One hundred mg/kg/day of p,p'-DDE significantly attenuated TP 0.1 mg/kg-induced increases in weights of seminal vesicles and muscles, and TP 1 mg/kg-induced increases in weights of ventral prostate, seminal vesicles and muscles, but did not affect the weight of these organs in either TP 16 mg/kg-treated or intact rats, demonstrating that the dose range of 0.1-1 mg/kg TP is suitable for reference androgen. Oral treatment with 100 mg/kg of MT increased the weights of ventral prostate, seminal vesicles and muscles as strongly as did subcutaneous injection of 1 mg/kg of TP. These findings demonstrate that the 5-day Hershberger assay using young mature as well as immature male rats is a sensitive and valid short-term screening method for the detection of chemicals interfering with AR-mediated mechanisms. To determine whether fenitrothion interferes with AR-mediated mechanisms in vivo, fenitrothion (0, 0.75, 1.5 or 3 mg/kg/day) was administered by gavage for 5 days to castrated rats for androgenicity, or to castrated rats treated with 1 mg/kg TP for antiandrogenicity. Treatment with fenitrothion had no adverse effects on clinical signs, body weight, or liver or kidney weights, but cholinesterase activities in the brain and erythrocytes were significantly suppressed by fenitrothion to, respectively, 77-81% and 66-67% of control levels. In the antiandrogenicity experiment, serum androgen levels of TP-treated, castrated rats did not differ among groups. Treatment with 100 mg/kg of p,p'-DDE as a positive control again significantly attenuated TP-induced increases in weights of the ventral prostate and seminal vesicles, while fenitrothion had no effect on the weights of any organs. In the androgenicity experiment, treatment with 100 mg/kg of MT significantly increased weights of ventral prostate, seminal vesicles and muscles, but fenitrothion had no effects on the weights of any of these organs. These findings yield no evidence that fenitrothion interferes with AR-mediated mechanisms in vivo, consistent with the result of several toxicological bioassays.

Immunotoxicological insignificance of fenitrothion in mice and rats.

Fenitrothion was administered orally to mice or rats in daily doses of up to 1/25 of the LD50 for 14 days, and numbers of splenic plaque-forming cells against sheep red blood cells (SRBC-PFC), one of the most common immune parameters, were measured. Splenic SRBC-PFC number was suppressed by fenitrothion only in rats which received 30 mg/kg body weight (bw) of the compound. Other immune parameters, including the arthus reaction, delayed-type hypersensitivity, and activities of macrophages and natural killer cells in rats, were not influenced by fenitrothion. Adrenal hyperfunction manifesting as increased organ weight and elevated plasma corticosterone level was noted along with strong cholinergic signs in rats which received 30 mg/kg bw of fenitrothion. At lower doses such as 3 or 0.3 mg/kg bw of fenitrothion, rats had no strong cholinergic signs, adrenal hyperfunction, or evidence of immunosuppression despite significant suppression of systemic cholinesterase (ChE) activities. In mice, no suppression of SRBC-PFC number or mixed lymphocyte reaction was noted even at the highest dose (40 mg/kg bw) of fenitrothion, at which significant suppression of systemic ChE activities but no cholinergic signs were noted. These findings strongly suggest that the immunosuppressive effect of fenitrothion noted in rats was due to systemic, potent cholinergic stress and that fenitrothion has no immunotoxicity in mice and rats.

Effect of an autoreactive T cell clone from (NZB x NZW)F1 mice on the production of anti-DNA antibodies in vivo and in vitro.

An autoreactive T lymphocyte clone, designated as F1C4 was established from an autoimmune mouse strain (NZB/NZW)F1. This clone proliferated in the presence of mitomycin C-treated splenic adherent cells (MMC-SAC) from syngeneic mice. This response was dependent on the numbers of MMC-SAC. The specificity of F1C4 for I-A was determined by an inhibition test carried out with monoclonal anti-Ia sera. Furthermore, the F1C4 cells did not exhibit alloreactivity in a proliferation assay and did not react to foreign antigens such as fetal calf serum (FCA) used in the culture medium. When F1C4 cells were cultured with autologous non-T cells in the absence of antigen, they strongly enhanced IgM class anti-ssDNA production from non-T cells of both young and old B/W F1 mice at appropriate cell numbers in vitro. Furthermore, the production of IgG class anti-ssDNA from non-T cells of old B/W F1 mice was also enhanced. The adoptive transfer of F1C4 cells enhanced the levels of both IgM and IgG anti-ssDNA antibodies in the serum of aged B/W F1 mice. Moreover, the serum levels of anti-ssDNA of IgG2a and IgG2b subclasses were readily enhanced by the transfer of F1C4 in vivo.

Endothelial cells in the rectal venous plexus. An electron microscopic study.

The endothelial cells of the rectal venous plexus were studied by electron microscope. The endothelial cytoplasm contained numerous specific granules, intracytoplasmic filaments, and 'anchoring filaments'. The present observations suggest that specific endothelial granules are formed in the Golgi apparatus. Thin 50-70 A and thick filaments 100-120 A were found in the endothelial cytoplasm. Also anchoring filaments between the plasma membrane of the endothelial cells and the underlying basement membrane (or subendothelial microfibrils) were present. The cytoplasmic filaments as well as the anchoring filaments are thought to be a support for the endothelial cells, functioning as a cytoskeleton.

Molecular and enzymatic properties of "cytochrome aa3"-type terminal oxidase derived from Nitrobacter agilis.

Cytochrome c oxidase (cytochrome aa3-type) [EC 1.9.3.1] was purified from Nitrobacter agilis to an electrophoretically homogeneous state and some of its properties were studied. The enzyme showed absorption peaks at 422, 598, and 840 nm in the oxidized form, and at 442 and 606 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 436 and 604 nm, and the latter peak had a shoulder at 599 nm. The enzyme possessed 1 mol of heme a and 1.6 g-atom of copper per 41,000 g, and was composed of two kinds of subunits of 51,000 and 31,000 daltons. These results show that the structurally minimal unit of the enzyme molecule is composed of one molecule each of the two subunits and contains 2 molecules of heme a and 2-3 atoms of copper. the enzyme rapidly oxidized ferrocytochromes c of several eukaryotes as well as N. agilis ferrocytochrome c-552. The reactions catalyzed by the enzyme were strongly inhibited by KCN. The reduction product of oxygen catalyzed by the enzyme was concluded to be water on the basis of the ratio of ferrocytochrome c oxidized to molecular oxygen consumed.

Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis.

Cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The T. novellus enzyme is composed of two kinds of subunits of 32,000 and 23,000 daltons and its minimum molecular weight is 55,000 on the basis of heme content and amino acid composition. The N. agilis enzyme also has two kinds of subunits of 40,000 and 27,000 daltons and its minimum molecular weight is 66,000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.

The terminal oxidase of Photobacterium phosphoreum. A novel cytochrome.

The terminal oxidase of Photobacterium phosphoreum has been purified to the electrophoretically homogeneous state and some of its properties have been studied. The enzyme catalyses oxidation of ascorbate in the presence of phenazine methosulphate or N,N,N',N'-tetramethyl-p-phenylenediamine. The reaction is inhibited by cyanide. Nitrite at comparatively high concentrations inhibits the enzyme, but the enzyme does not catalyse nitrite reduction with ascorbate plus the electron mediator as the electron donor. The enzyme shows the absorption peaks at 632, 565, 534 and 436 nm in the reduced form. It has two kinds of haems: protohaem and haem d. Namely, the enzyme is a 'cytochrome bd'-type oxidase; a novel cytochrome.