Inhalation toxicity of methylisocyanate: assessment of germ cell mutagenicity and reproductive effects in rats.

Adult male Wistar rats were exposed to methylisocyanate (MIC, 3.2 mg/l, single inhalation exposure for 8 min under static condition) or ethyl methanesulphonate (EMS, 150 mg/kg, single ip dose) for the assessment of germ cell mutagenicity and reproductive effects. Sequential matings of treated males with normal females on days 1-7, 8-14 and 15-21 post-exposure did not indicate any induction of dominant lethal mutation (increased frequency of preimplantation losses and early fetal deaths) by MIC but it was significantly induced by EMS as compared to respective controls. Males, necropsied after 21 days of exposure, showed no effect of MIC on epididymal sperm density and morphology. EMS also had no effect on sperm density but it significantly induced morphological abnormalities in sperm as compared to untreated controls. There was an acute and transitional reduction in reproductive performance (10-21%) of MIC-exposed males during days 1-14 post-exposure followed by recovery to the normal level during days 15-21 post-exposure. The progeny of MIC-exposed males was also normal in terms of litter size, litter weight, neonatal survival and body weight gain in litters up to 10 days post-partem. It is concluded with the evidence at hand that the observed failure of MIC to cause germ cell mutagenicity is related to its poor biodistribution to the target site(s) and a transient reduction in the reproductive performance of MIC-exposed males is a result of general stress and disconsolate copulation.     

Activation of mineralocorticoid agonist and antagonist specific receptors from rat kidney

The kinetics of saturation, as well as of denaturation, confirm the existence of two distinct mineralocorticoid receptor populations one each for the agonist aldosterone (MR2) and the antagonist RU 26752 (MR3) in rat kidney. Receptor activation in vitro was dependent upon the buffer, progressed just as well in the presence of the agonist and the antagonist, and was inhibited by molybdate. These necessitate a reassessment of both the importance of receptor activation in vitro and its possible contribution to hormone action in vivo.     

Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

Summary A total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1^*1, GST1^*2, and GST1^*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1^*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 M, respectively.     

Circulating concentrations of thyroid hormones in pregnant camels (Camelus dromedarius )

Blood samples from 16 female camels were collected at monthly intervals commencing from 60 d post. breeding until the last month of gestation. Two camels failed to conceive and two had unnoticed abortions. The average gestation period was 398+/-13 and 372+/-11 in camels bearing male and female fetus, respectively, with an overall mean of 383+/-9 d. Sera were analyzed for thyroxine (T(4)) and triiodothyronine (T(3)) by radioimmunoassay. Mean T(4) and T(3) concentrations varied from 76 to 116 ng/ml and 0.73 to 1.32 ng/ml, respectively, during various stages of gestation. In general, the T(4) and T(3) levels were higher during early pregnancy, with lowest values in the tenth month. T(4):T(3) ratio showed minor, nonsignificant fluctuations. Age of dam of sex of fetus had no effect on hormone levels. Similarly, hormone levels were not affected by failure of conception or by abortion.     

The role of microtubules in platelet secretory release

The role of microtubules in platelet aggregation and secretion has been analyzed using platelets permeabilized with digitonin and monoclonal antibodies to alpha (DM1A) and beta (DM1B) subunits of tubulin. Permeabilized platelets were able to undergo aggregation and secretory release. However, threshold doses of agonists capable of eliciting a second wave of aggregation and the platelet release reaction were higher than in control platelets exposed to dimethyl sulfoxide, the solvent for digitonin. Both antibodies to alpha and beta tubulin caused a further increase in the threshold concentration of agonists and inhibited the secretory release of permeabilized platelets, but were ineffective using intact platelets. Neither monoclonal antibody inhibited polymerization or depolymerization of platelet tubulin in vitro. Antibodies to platelet actin and myosin also exhibited an inhibitory activity on platelet aggregation albeit less severe than that observed with the antibodies to alpha and beta tubulin. There was evidence of an interaction between DM1A and DM1B and the antibodies to actin and myosin. The interaction of platelet tubulin and myosin was investigated by two different methods. (1) Coprecipitation of the proteins at low ionic strength at which tubulin by itself did not precipitate and (2) affinity chromatography on columns of immobilized myosin. Tubulin freed of its associated proteins (MAPs) by phosphocellulose chromatography bound to myosin in a molar ratio which approached 2. Platelet actin competed with tubulin for 1 binding site on the myosin molecule. MAPs also reduced the binding stoichiometry of tubulin/myosin. Treatment of microtubule protein with p-chloromercuribenzoate or colchicine did not influence its binding to myosin. DM1A and DM1B inhibited the interaction of tubulin and myosin. This effect could also be demonstrated by reaction of electrophoretic transblots of extracted platelet tubulin with the respective proteins. We interpret these results as evidence for an interference of the two monoclonal antibodies to the tubulin subunits (DM1A and DM1B) with the translocation of microtubule protein from its submembranous site to a more central one during the activation process.     

Binding properties of diethylcarbamazine

Diethylcarbamazine (DEC) reacted with liver cell plasma membrane of rodent hosts-cotton rat, albino rat and Mastomys natalensis exhibiting the presence of both saturable and unsaturable components. The presence of lectins or sugar derivatives did not affect the binding significantly. The drug showed similar binding pattern with serum but the saturation was reached at a much lower concentration of the ligand. Data obtained with a variety of macromolecules, particularly with the homopolymers of amino acids indicate that DEC does not require any specific constituent of the membrane for binding. The nonspecific nature of DEC binding does not provide any convincing clue for the accumulation of microfilariae specifically in the liver following the drug treatment.     

A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro

Summary Many research efforts require the accurate determination of cell density in vitro. However, physical cell counting is inaccurate, time-intensive and requires removal of the cells from their growth environment, thereby introducing a host of potential artifacts. The current studies document a very simple method of determining cell density in microtiter wells via DNA-enhanced fluorescence. Fixed cells are stained with the A-T intercalating DNA stains DAPI or Hoechst 33342 and then fluorescence is quantified in a plate fluorometer. Fluorescence is shown to be linearly related to cell density as determined by two physical counting methods. The validity of the method is established in determining serum-stimulated growth of smooth muscle cells and in mitogen-induced growth of endothelial cells. The fixed cells can be stored for prolonged periods, thus allowing time-course proliferation assays without interassay variations. The fixed cells are also suitable for determinations of antigens of interest by ELISA. This method is potentially valuable in many in vitro systems where the quantification of cell density and proliferation is necessary. This work supported in part by NIH Cardiovascular Training Grant HL07423 and a grant from the American Federation for Aging Research to T. M. and HL35724 to B. W. EDITOR’S STATEMENT The technique described in this paper represents an approach to quantifying cell density in adherent monolayers of cultured cells in microtiter wells that is rapid and simple and does not require radioisotopes or removal of cells.     

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins.

A cell fixation and permeabilization procedure consisting of sequential paraformaldehyde and methanol was evaluated and found suitable for concomitant flow cytometric quantification of total cellular DNA, immunofluorescence measurements of cell surface proteins, and immunofluorescence measurements of intracellular proteins. Paraformaldehyde/methanol-fixed cells exhibited significantly greater intracellular antitubulin immunofluorescence than cells fixed with paraformaldehyde or methanol alone (p less than 0.002) and significantly greater intracellular antitubulin immunofluorescence than cells fixed with methanol followed by paraformaldehyde (p less than 0.006). With paraformaldehyde/methanol fixation, cell morphology was well preserved and forward and right angle light scatter properties were sufficiently well maintained to permit gating on these parameters. Cell surface marker staining with fluorescent anti-leukocyte antibodies was unaffected by fixation with paraformaldehyde/methanol. Paraformaldehyde effects on the intensity of DNA staining with propidium iodide were dependent on paraformaldehyde concentration and fixation temperature; these effects were least pronounced at low paraformaldehyde concentrations (0.25% or less), and at temperatures lower than 37 degrees C. Paraformaldehyde fixation may result in differences in propidium iodide staining of DNA in some diploid cells, which may produce small spurious aneuploid peaks in normal peripheral blood leukocytes. Paraformaldehyde fixation also produces an apparent increase in the DNA index of aneuploid cell populations in comparison with methanol fixation, particularly when the DNA index exceeds 1.5. Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors.     

Effects of cyclic AMP and butyrate on cell cycle,DNA, RNA,and purine synthesis of cultured astrocytes

Dibutyryl cyclic monophosphate (dBcAMP) has been shown to inhibit growth, and alter the morphology of astrocytes. However, the potential contribution of its hydrolytic product, butyrate, in inducing some of the changes that have been attributed to dBcAMP, is not clear. DNA, RNA, and purine synthesis were therefore studied in primary astrocyte cultures after 24 hours of exposure to varying concentrations of butyrate, dBcAMP, and agents that increase intracellular cAMP levels. Progression of cells through cell cycle was also studied by flow cytometry. Dibutyryl cAMP partially arrested cells in Go/G1 phase of cell cycle while sodium butyrate increased the percentage population of cells in G2/M phase. DNA synthesis and de novo purine synthesis were inhibited after treatment with dBcAMP, sodium butyrate, and various drugs that increase intracellular cAMP levels. RNA synthesis was increased with cAMP but was not affected by sodium butyrate. Our study shows that at millimolar concentrations, butyrate is capable of altering the cell cycle and inhibiting DNA synthesis in primary astrocyte cultures, in a manner that is similar although not identical to the effects of dBcAMP.     

Antimutagenic and antitumorigenic activities of nordihydroguaiaretic acid.

Nordihydroguaiaretic acid (NDGA), which occurs in the resinous exudates of many plants is used as an antioxidant in fats and oils. In this study we show that NDGA inhibited the mutagenicity of methyl methanesulfonate, benzo[a]pyrene (BP), 2-aminofluorene, and aflatoxin B1 in Salmonella typhimurium strain TA100 or TA98 in the absence and presence of rat hepatic microsomal activation system. The addition of NDGA during and after nitrosation of methylurea (MU) resulted in a dose-dependent inhibition of mutagenicity induced by nitrosation products of MU. In a two-stage skin tumorigenesis protocol using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiating agent followed by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) as tumor promoter, pretreatment of animals with NDGA prior to DMBA application, afforded significant protection against skin tumorigenicity in female SENCAR mice. In additional studies, skin application of NDGA also inhibited the binding of topically applied [3H]BP and [3H]DMBA to epidermal DNA. When assessed in the anti-tumor promotion protocol, pretreatment of animals with NDGA before each application of TPA in DMBA-initiated mouse skin, resulted in 72% decrease in the total number of tumors when compared to non-NDGA pretreated animals. The possible mechanism(s) of the antimutagenic and anti-tumorigenic activities may be due to the multiple effects of NDGA as inhibitor of the carcinogen metabolism and DNA-adduct formation, scavenger of carcinogen free radicals, and as inhibitor of TPA-induced ornithine decarboxylase activity.