Cytogenetic studies of three triazine herbicides. II. In vivo micronucleus studies in mouse bone marrow

Atrazine, simazine, and cyanazine are widely used preemergence and postemergence triazine herbicides that have made their way into the potable water supply of many agricultural communities. Although there are several contradictory genotoxicity studies in the literature, our previous in vitro studies with human lymphocytes showed that atrazine, simazine, and cyanazine did not induce sister chromatid exchanges (SCEs) or chromosome aberrations (CAs) up to the limits of solubility in aqueous medium using 0.5% dimethyl sulfoxide. To expand upon these results and to ensure that our in vitro findings could be replicated in an in vivo system, mice were treated with each triazine by two intraperitoneal injections, 24h apart. The animals were sacrificed and the bone marrow removed for micronucleus (MN) analysis, 24h after the last injection. Two to four independent trials were performed for MN analysis in polychromatic erythrocytes, and in some trials the spleen was removed, cultured, and analyzed for SCEs and CAs. None of the triazines investigated induced MN in the bone marrow, even at doses that caused significant bone marrow suppression and/or death. These results indicate that atrazine, simazine, and cyanazine are not genotoxic as measured by the bone marrow MN assay in mice following high dose exposures.     

Genotoxicity studies of three triazine herbicides: in vivo studies using the alkaline single cell gel (SCG) assay

Triazine herbicides are prevalent contaminants of groundwater in the agricultural regions of the United States. The literature on the genotoxicity of triazines is rife with conflicting data, though the general tendency is for most studies to report negative results. In order to investigate further the genotoxicity of triazines, we exposed mice to triazines by intraperitoneal injection up to the maximum tolerated doses. About 24h later, blood was removed, and the leukocytes subjected to DNA damage analysis using the alkaline single cell gel electrophoresis assay (SCG), one of the most sensitive DNA damage assays available. Our results indicate that atrazine induced a small dose-related increase in DNA damage. Simazine did not induce any dose-related increase in DNA damage. Cyanazine induced a marginal increase in DNA damage with dose, but no individual dose was significantly increased compared to the control. These results indicate that these triazines, even at extremely high concentrations, have only marginal DNA-damaging activity in vivo in mouse leukocytes.     

In vitro studies on the effects of herbicides on the growth of rhizobia

AIMS: To study the possible adverse effect of herbicides on nodulation and nitrogen fixation in legumes by affecting the nitrogen-fixing rhizobia. METHODS AND RESULTS: Experiments were conducted to study the effect of four herbicides (terbutryn/terbuthylazine, trietazine/simazine, prometryn and bentazone) on the growth of nitrogen-fixing pea rhizobia (Rhizobium leguminosarum) in vitro by measuring optical density. Terbutryn/terbuthylazine, trietazine/simazine and prometryn had an adverse effect on the growth of rhizobia whereas bentazone was safe to rhizobia. CONCLUSIONS: The above herbicides could be used in pea at the recommended rates. SIGNIFICANCE AND IMPACT OF THE STUDY: The adverse effects of herbicides on rhizobia were observed at concentrations not normally expected to occur under field conditions. Further, previously observed adverse effects of these herbicides on nodulation and nitrogen fixation of peas were, possibly, not due to their effects in rhizobia but to their adverse effects on the plant growth itself.     

In vitro anticryptosporidial activity of dinitroaniline herbicides

Abstract Despite the evaluation of over 100 antimicrobial drugs, the diarrheal disease cryptosporidiosis has remained refractory to treatment. We report the evaluation of five dinitroaniline herbicides including trifluralin, profluralin, nitralin, pendimethalin, and fluchloralin for anticryptosporidial activity in an in vitro cultivation model of Cryptosporidium parvum . All five compounds exhibited significant anticryptosporidial activities with no corresponding evidence of toxicity. The most active compound was pendimethalin with an IC₅₀ of 0.19 μM while nitralin was the least active with an IC₅₀ of 4.5 μM. These compounds should be evaluated further in an animal model of cryptosporidiosis.     

The binding of triazine herbicides to the photosynthetic reaction center of Rhodopseudomonas viridis. Energy minimization studies.

The binding of six herbicides of the triazine family to the photosynthetic reaction center of Rhodopseudomonas viridis was investigated with energy-minimization techniques, in order to correlate experimental with calculated data. The inhibitors were modeled in the active site according to the X-ray structure analysis of the complex formed between the triazine terbutryn (2-ethylamino-4-t-butylamino-6-methylthio-s-triazine) and the reaction center of R. viridis [Michel, H., Epp. O. & Deisenhofer, J. (1986) EMBO J. 5, 2445-2451]. 40 different energy minimizations were carried out with varying cutoff radii, partial charges on inhibitor atoms and dielectric constants, i.e. 10 different combinations of these were tested. The impact of these parameters on the calculated binding and interaction energy was either examined for all protein/triazine complexes or, in the case of the dielectric constant, a smaller sample was used. The calculated energies are dominated by van der Waals interactions, which change by up to 20% when extending the cutoff radius from 0.8 nm to 1.5 nm. The use of uniform or distance-dependent dielectric constant or partial charges on the inhibitor atoms does not severely influence the resulting structures, but shows a great impact on the calculated energies. In the two groups of triazines, each containing three inhibitors with methoxy or methylthio substituents, correlations of biological and calculated data were found quite often, but only once with all six triazines. The energy-minimized structures were compared and analysed. A third hydrogen bond, not seen in the X-ray analysis of the reaction center/tertubryn complex, was found between the t-butylamino moiety of terbutryn (and equivalent moieties in the other triazines) and the carbonyl oxygen of TyrL222.     

In vitro morphogenetic studies on three apomictic species of Garcinia

Comparison of morphogenetic potential of three important Indian species of Garcinia??G. indica, G. cambogia and G. xanthochymus has been reported. Apomictic seeds of G. indica were found to be morphogenetically most potential followed by G. cambogia. The explants of G. xanthochymus were highly recalcitrant towards in vitro conditions and failed to induce adventitious buds on any of the media tested. High frequency direct shoot bud differentiation was induced in aseptic seed cultures of G. indica and G. cambogia on MS medium supplemented with cytokinins (BAP, kinetin or TDZ). Amongst the three cytokinins tested, TDZ (0.1?C0.5???M) was most effective for adventitious bud differentiation in both G. indica and G. cambogia, however, the proliferating buds failed to elongate. Substantial number of buds induced on BAP supplemented media elongated into shoots after subculture on elongation medium. Addition of NAA along with cytokinins in the induction medium enhanced callusing without improvement in bud induction response. The induced adventitious buds were elongated on MS basal medium containing 0.2% activated charcoal. Direct rooting was achieved in both G. indica and G. cambogia on auxin supplemented media with best response at 10???M IBA concentration in both the species. The in vitro raised plantlets showed 90% survival in the field when transferred after hardening and acclimatization.     

Cytogenetic studies in mammalian cells after treatment with insect pathogenic viruses [Baculoviridae]. I.In vivo studies with rodents

No consistent deleterious effects directly attributable to the nuclear polyhedrosis virus ofMamestra brassicae (L.),Autographa californica (Speyer) and the granulosis virus ofLaspeyresia pomonella (L.) were observed, when bone marrow cells of Chinese hamsters and NMRI mice were examined for chromosomal aberrations or sister chromatic exchanges. The animals were exposed to thein vivo andin vitro produced viruses acutely or chronicaly (90 resp. 97 days) by oral gavage or intraperitoneal injection. The treated animals neither showed signs of health disturbances nor abnormal behaviour nor changes in body weight in comparison to the controls.     

In vitro studies of age-associated diseases.

We have carried out studies on cultured human fibroblasts in an attempt to trace the origins of age-dependent disorders to the cellular and molecular levels. Three interrelated areas are discussed. First, skin donors with diabetes mellitus (a disease complex that features inappropriate hyperglycemia) produce cultured fibroblasts with a moderate reduction in growth capacity, while two inherited disorders of inappropriate hyperglycemia and premature aging, progeria and Werner syndrome, yield fibroblast cultures with more severely impaired growth capacity. Second, there is a decreased response of progeria level and donor age; evidence is presented that this defective hormone responsiveness in aging cells may reside at the hormone receptor on the surface membrane, the cyclic AMP system, the intracellular enzymatic machinery, or all of these sites. Third, tissue factor, a procoagulant that activates the extrinsic clotting mechanism, is more abundant in cells from the premature aging syndromes of progeria and Werner syndrome. Fibroblast aging in vitro may help to explain various concomitants of normal aging and diabetes mellitus including cell dropout, impairment of hormone responsiveness, and increased atherothrombosis.     

Invertebrate biodiversity in maize following withdrawal of triazine herbicides

Responses of key invertebrates within Farm Scale Evaluations (FSEs) of maize reflected advantageous effects for weeds under genetically modified herbicide-tolerant (GMHT) management. Triazine herbicides constitute the main weed control in current conventional systems, but will be withdrawn under future EU guidelines. Here, we reappraise FSE data to predict effects of this withdrawal on invertebrate biodiversity under alternative management scenarios. Invertebrate indicators showed remarkably consistent and sensitive responses to weed abundance. Their numbers were consistently reduced by atrazine used prior to seedling emergence, but at reduced levels compared to similar observations for weeds. Large treatment effects were, therefore, maintained for invertebrates when comparing other conventional herbicide treatments with GMHT, despite reduced differences in weed abundance. In particular, benefits of GMHT remained under comparisons with best estimates of future conventional management without triazines. Pitfall trapped Collembola, seed-feeding carabids and a linyphiid spider followed closely trends for weeds and may, therefore, prove useful for modelling wider biodiversity effects of herbicides. Weaker responses to triazines applied later in the season, at times closer to the activity and capture of invertebrates, suggest an absence of substantial direct effects. Contrary responses for some suction-sampled Collembola and the carabid Loricera pilicornis were probably caused by a direct deleterious effect of triazines.     

Cytogenetic studies in mammalian cells after treatment with insect pathogenic viruses [Baculoviridae]. II.In vitro studies with mammalian cell lines

Mammalian cell-lines from Chinese hamster, Indian muntjac and mouse were inoculated with infectious supernatant ofAutographa californica (Speyer) nuclear polyhedrosis virus (Ac NPV) replicated inMamestra brassicae (L.) cell cultures (IZD-Mb-0503). There was no adverse effect on cell proliferation, nor was a cytopathogenic effect (CPE) induced in such cultures. Cytogenetic data indicate that uptake ofAc-NPVs into the cytoplasm of mammalian cells, as shown by an electron microscopic study, induced neither numerical or structural chromosome aberrations nor sister chromatid exchange (SCE) events.     

II. In vitro studies of antibacterial activity

One hundred and forty isolates of beta-hemolytic streptococcus cultured from patients with clinical pharyngitis were studied by disc diffusion for antibiotic sensitivity to lincomycin, erythromycin, cephalexin and penicillin and by agar dilution to cephalexin and penicillin. All isolates were sensitive to ≤ 0.1 μg./ml. penicillin and ≤ 1.56 μg./ml. cephalexin. The disc-diffusion test was reliable in predicting the sensitivities in vitro. One strain of group A betahemolytic streptococcus was resistant to erythromycin by disc diffusion. When compared to Lancefield grouping 18% of strains were incorrectly identified as group A by the bacitracin-disc test. Cephalexin was uniformly effective in vitro in inhibiting beta-hemolytic streptococci and the 30 μg. cephalexin disc was reliable in predicting these sensitivities.     

In vitro studies of membrane protein folding.

The study of membrane protein folding is a new and challenging research field. Consequently, there are few direct studies on the in vitro folding of membrane proteins. This review covers work aimed at understanding folding mechanisms and the intermolecular forces that drive the folding of integral membrane proteins. We discuss the kinetic and thermodynamic studies that have been undertaken. Our review also draws on closely related research, mainly from purification studies of functional membrane proteins, and gives an overview of some of the successful methods. A brief survey is also given of the large body of mutagenesis and fragment work on membrane proteins, as this too has relevance to the folding problem. It is noticeable that the choice of solubilizing detergents and lipids can determine the success of the method, and indeed it appears that particular lipid properties can be used to control the rate and efficiency of folding. This has important ramifications for much in vitro folding work in that it aids our understanding of how to obtain and handle folded, functional protein. With this in mind, we also cover some relevant properties of model, lipid-bilayer systems.     

In vitro studies on transplantation immunity. I. MIF production by sensitive lymphocytes in mice

Sensitized lymphocytes from mice immunized with skin homografts produce migration inhibitory factor upon incubation with lymphocytes (antigen) from the sensitizing strain. The MIF is produced within 14 hr following incubation of sensitized lymphocytes and antigen. In this reaction, antigenic specificity is a prerequisite for MIF production; however, the action of MIF transcends the strain barrier. Also, MIF produced in homograft reactions in mice inhibited the migration of peritoneal cells from normal guinea pigs. Finally, lymphocytes from mice bearing skin homografts do not develop the capacity to produce MIF prior to the rejection of the sensitizing skin grafts.