Evaluation of hexamethylphosphoramide for gene mutations in Salmonella typhimurium using plate incorporation, preincubation, and suspension assays.

Hexamethylphosphoramide (HMPA), a potent rat nasal carcinogen by inhalation, and three of its metabolites, pentamethylphosphoramide (PMPA), trimethylphosphoramide (TriMPA), and formaldehyde (HCHO), were assessed in Salmonella typhimurium gene mutation assays using various protocols, including plate incorporation, preincubation and suspension assays. HMPA (tested up to 15,000 micrograms/plate) was not mutagenic in plate incorporation or preincubation assays with or without metabolic activation. HCHO was mutagenic in the plate incorporation and preincubation assays (tested up to 150 micrograms/plate). In suspension assays, however, HMPA (tested up to 40 mg/ml), PMPA (up to 44 mg/ml) and HCHO (up to 45 micrograms/ml), but not TriMPA (up to 29 mg/ml), were mutagenic. HMPA and PMPA were positive only with activation. HMPA's mutagenicity was optimized using a relatively high level of rat liver S9 protein (3.5 mg/plate) in the metabolic activation mixture. Semicarbazide, an HCHO trapping agent, added at concentrations up to 167 micrograms/ml, markedly inhibited the mutagenic activities of HMPA and PMPA suggesting that HCHO generation may play a role in their mutagenicity. These studies show that HMPA is mutagenic in a modified Salmonella typhimurium reverse mutation assay with metabolic activation. Successive N-demethylation of HMPA eventually eliminates the mutagenic activity which further suggests that HMPA's mutagenic activity is related to the release of HCHO.

Evaluation of benomyl and carbendazim in the in vivo aneuploidy/micronucleus assay in BDF1 mouse bone marrow.

Benomyl and its active metabolite carbendazim were investigated in BDF1 mouse bone marrow to establish whether micronuclei induced by these fungicides are caused by clastogenic or aneugenic events. Micronuclei were evaluated for kinetochores using immunofluorescent antikinetochore antibodies. Kinetochore positive (K+) micronuclei are likely to arise from chromosome loss since they presumably contain intact kinetochores and are indicative of aneuploidy. Conversely, kinetochore negative (K-) micronuclei are mostly likely to contain acentric chromosome fragments arising primarily from clastogenic damage. Benomyl and carbendazim were administered as single oral doses of 0.3, 8.6 or 17.2 mmol/kg (for benomyl, equivalent to 100, 2500 or 5000 mg/kg; for carbendazim, equivalent to 66, 1646 or 3293 mg/kg). Both compounds were positive in the micronucleus test at doses of 8.6 and 17.2 mmol/kg, and an average of 82% (benomyl) and 87% (carbendazim) of the total micronucleated polychromatic erythrocytes were K+. No effects were seen with either fungicide at 0.3 mmol/kg. These results are analogous to findings with known aneugens such as vincristine but are in contrast to results with classical clastogens such as cyclophosphamide. Thus, benomyl and carbendazim induce micronuclei in mouse bone marrow cells primarily through an aneugenic mechanism.

Evaluation of carbendazim for gene mutations in the Salmonella/Ames plate-incorporation assay: the role of aminophenazine impurities.

Benomyl (methyl [1-[(butylamino)carbonyl]-1H-benzimidazol-2- yl]carbamate) and its major metabolite carbendazim (methyl 2-benzimidazolecarbamate) are major agricultural systemic fungicides. These compounds inhibit fungal microtubular function and thereby cause nondisjunction of chromosomes at cell division. Several investigators have proposed that these compounds can also cause gene mutations (base-pair substitutions). In this laboratory, no mutagenic activity was observed with either benomyl (analytical grade) or Benlate (samples tested up to 500 and 1200 micrograms/plate, respectively, the limit of cytotoxicity) in the Salmonella/Ames plate-incorporation test in either base-pair substitution (TA100 and TA1535) or frameshift-sensitive (TA98 and TA1537) strains with or without S9 metabolic activation. However, some carbendazim preparations caused mutations in frameshift-sensitive strains at very high concentrations (> or = 5000 micrograms/plate) with metabolic activation. The mutagenic activity was not due to the major carbendazim metabolite, methyl (5-hydroxy-1H-benzimidazol-2-yl)carbamate (5-OH MBC), since 5-OH MBC was not mutagenic with (up to 20,000 micrograms/plate) or without (up to 16,000 micrograms/plate) activation. Subsequently, two highly mutagenic contaminants, 2,3-diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP) were detected in mutagenic carbendazim samples. In those samples, DAP and AHP contaminant levels ranged as high as 46.5 and 11.6 ppm, respectively. No evidence of mutagenicity could be detected in preparations in which the DAP content was < 1.8 ppm. The mutagenic activity of these two contaminants was further investigated in strain TA98. Without activation, DAP and AHP were positive at test concentrations as low as 5 and 10 micrograms/plate, respectively. In the presence of S9, mutations were detected at much lower concentrations (beginning at 0.025 and 0.05 microgram/plate, respectively). These results indicate that carbendazim samples containing DAP or AHP at levels as low as 5 or 10 ppm, respectively, would be positive in the Salmonella/Ames test with activation when tested at 5000 micrograms/plate. Purified carbendazim is not mutagenic.

Mechanical wounding induces the formation of extensive coated membranes in giant algal cells.

Mechanically wounding giant cells of Boergesenia forbesii induces the formation of bristle-coated, plasma-membrane invaginations (coated pits) and coated vesicles, easily providing a plentiful source of coated membranes in a clean cellular system unencumbered by other tissues. Contractions evoked by wounding partition the cytoplasm into hundreds of spherical protoplasts with approximately 40 percent less total plasma-membrane surface area than the original cell. Ferritin labeling and the appearance of numerous large coated pits and vesicles at the peak period of contraction indicate that these organelles play a role in extensive endocytosis of the plasma membrane.

A comparative study of the respiratory responses to bronchoactive agents in rhesus and cynomolgus monkeys.

Respiratory responses to a variety of known bronchoactive agents were compared in anesthetized rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) monkeys. Administration of 0.1 to 1.0% histamine aerosols resulted in an increase in airway resistance of 50 to 200% and a decrease in lung compliance of 30 to 80%. Aerosols of prostaglandin E2 (1 mg/ml), terbutaline (10 mg/ml), and isoproterenol (10 mg/ml) or iv aminophylline (up to 7.0 mg/kg) administered concomitantly with histamine produced a transient reversal of the histamine-induced changes in both species. Since the rhesus and cynomolgus monkeys responded in a comparable manner to these bronchodilator agents, the cynomolgus monkey appears to be an additional valuable model for the evaluation of potential bronchoactive compounds.

Respiratory responses to Ascaris antigen in rhesus and cynomolgus monkeys.

Responses to Ascaris antigen were evaluated in rhesus and cynomolgus monkeys. Of 19 cynomolgus monkeys tested, 15 were found to have cutaneous reactivity to Ascaris: 13 of these responded to Ascaris aerosols with changes in respiratory function including an increase in respiration rate, decrease in tidal volume and peak expiratory flow rate, decrease in dynamic compliance, and an increase in resistance. Four of the six rhesus monkeys studied with cutaneous reactivity to Ascaris responded to Ascaris aerosols: the changes in respiratory function observed after the Ascaris challenge in these monkeys were similar to those observed in the cynomolgus monkeys. Responses to repetitive Ascaris challenges were obtained in monkeys of both species with a recovery period of 30 to 60 min between challenges. In monkeys of both species that reacted to Ascaris aerosols, blood pressure was elevated transiently; changes in heart rate were variable. Changes in cardiovascular measurements were not observed in monkeys that did not respond to Ascaris aerosols. Thus, it appears that the cynomolgus monkey responds to an aerosol Ascaris challenge in a manner similar to the rhesus monkey and is an additional suitable model for the study of allergic respiratory responses.

Delta-9-tetrahydrocannabinol on isolated human bronchioles.

Experiments were conducted to investigate the bronchodilating action of delta-9-tetrahydrocannabinol (THC) using normal human lung tissue obtained post mortem. Isolated rings of human bronchioles contracted to histamine, carbachol, and prostaglandin F(2a)(PGF(2a)) and relaxed to isoproterenol. THC (5 X 10(-4)M) did not alter the responses of the bronchial rings to histamine, whereas methapyrilene (10(-6)M) antagonized these responses. Atropine (2 X 10(-6)M) had a highly significant effect on the responses of the bronchioles to carbachol: there was also a significant effect of THC (5 X 10(-4)M), but to a much lesser extent than atropine. Propranolol (10(-6)M) pretreatment significantly antagonized the relaxant responses of the bronchioles to isoproternol: THC antagonized these responses to a smaller degree. Incubation with THC did not cause relaxation of resting tissues or tissues in which a spasm had been induced. These data suggest that THC does not have significant direct effects in human bronchial smooth muscle and that bronchoactivity observed in vivo is likely to be of a nondirect or central origin.

L-Glutamic acid as a mediator of sexual morphogenesis in Volvox capensis.

In Volvox capensis the development of sexual individuals is in response to low concentrations (68 nM) of L-glutamic acid rather than to such species-specific glycoproteins as have been isolated in Volvox carteri or are believed to exist in a number of other species. V. capensis grows equally as well in light and in darkness in a medium supplemented with sodium acetate; however, L-glutamic acid is active as an inducer of the sexual form only in populations grown in the light. The site of action of L-glutamic acid and its biochemical role in the sexual response are unknown. Attempts to induce the sexual response by using the other L-amino acids, various analogs of glutamic acid, compounds of similar structure (e.g., gamma-aminobutyric acid), and intermediates of biochemical pathways known to involve L-glutamic acid (e.g., alpha-ketoglutarate or pyroglutamic acid) have been unsuccessful. L-Glutamic acid is produced by V. capensis as a natural product of the digestion of the glycoproteinaceous parental matrix at the time young spheroids escape. As a population increases, so does the level of L-glutamic acid produced at each succeeding generation until the threshold of sensitivity is reached and the induction of sexual forms is effected. This serves as a mechanism for ensuring the production of sexual spheroids and their zygotes, the only phase in the life cycle resistant to drying. Thus, Volvox is especially adapted to an existence in ephemeral pools of water resulting from seasonal rains.