Genotoxic effect of 2,4-dichlorophenoxy acetic acid and its metabolite 2,4-dichlorophenol in mouse.

The cytogenetic effect of 2,4-dichlorophenoxy acetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (2,4-DCP) was studied in bone-marrow, germ cells and sperm head abnormalities in the treated mice. Swiss mice were treated orally by gavage with 2,4-D at 1.7, 3.3 and 33 mg kg(-1)BW (1/200, 1/100 and 1/10 of LD(50)). 2,4-DCP was intraperitoneally (i.p.) injected at 36, 72 and 180 mg kg(-1)BW (1/10, 1/5, 1/2 of LD(50)). A significant increase in the percentage of chromosome aberrations in bone-marrow and spermatocyte cells was observed after oral administration of 2,4-D at 3.3 mg kg(-1)BW for three and five consecutive days. This percentage increased and reached 10.8+/-0.87 (P<0.01) in bone-marrow and 9.8+/-0.45 (P<0.01) in spermatocyte cells after oral administration of 2,4-D at 33 mg kg(-1)BW for 24 h. This percentage was, however, lower than that induced in bone-marrow and spermatocyte cells by mitomycin C (positive control). 2,4-D induced a dose-dependent increase in the percentage of sperm head abnormalities. The genotoxic effect of 2,4-DCP is weaker than that of 2,4-D, as indicated by the lower percentage of the induced chromosome aberrations (in bone-marrow and spermatocyte cells) and sperm head abnormalities. Only the highest tested concentration of 2,4-DCP (180 mg kg(-1)BW, 1/2 LD(50)) induced a significant percentage of chromosome aberrations and sperm head abnormalities after i.p. injection. The obtained results indicate that 2,4-D is genotoxic in mice in vivo under the conditions tested. Hence, more care should be given to the application of 2,4-D on edible crops since repeated uses may underlie a health hazard.

Spectrodensitometric determination of clorazepate dipotassium, primidone and chlorzoxazone each in presence of its degradation product.

The present work describes a quantitative thin layer procedure for estimating primidone, clorazepate dipotassium and chlorzoxazone in bulk powders and in dosage forms, each in the presence of its degradation product. The method consists of dissolving the drug in ethanol (for primidone), or methanol (for clorazepate dipotassium and chlorzoxazone) and then spotting this solution on a thin layer of silica gel G254. Quantitation is achieved by comparing the areas under the peaks obtained from scanning the thin layer chromatographic plates in a spectrodensitometer.

Prevention of brefeldin A-induced resistance to teniposide by the proteasome inhibitor MG-132: involvement of NF-kappaB activation in drug resistance.

Brefeldin A, an agent that disrupts protein transport from the endoplasmic reticulum to the Golgi, induces the expression of GRP78 and the activation of nuclear factor (NF)-kappaB in cells. Treatment of cells with brefeldin A causes the development of resistance to topoisomerase II-directed agents, such as etoposide and doxorubicin. In this study, we show that treatment of EMT6 mouse mammary tumor cells with brefeldin A strongly induces GRP78 mRNA (8.5-fold) and resistance to teniposide (VM26). Treatment with okadaic acid causes a minor increase in GRP78 mRNA (2.1-fold) yet still induces resistance to VM26 as effectively as brefeldin A. In contrast, cells treated with castanospermine show a moderate increase in GRP78 mRNA (3.9-fold) but no resistance to VM26. These data imply that GRP78 induction does not mediate the development of drug resistance. An alternative mechanism of drug resistance may involve activation of the transcription factor, NF-kappaB, and we show that both brefeldin A and okadaic acid activate NF-kappaB in EMT6 cells. Furthermore, we demonstrate that treatment with the proteasome inhibitor MG-132 blocks the activation of NF-kappaB and prevents the development of resistance to VM26 induced by brefeldin A. Collectively, these results suggest that the resistance to VM26 in EMT6 cells treated with brefeldin A is mediated by the activation of NF-kappaB rather than the induction of GRP78. Our results also suggest that inhibition of NF-kappaB activation in tumor cells may increase the efficacy of topoisomerase II-directed agents in chemotherapy.

Cytogenetic effect of some insecticides in mouse spleen.

Several insecticides were tested for their ability to induce chromosomal aberrations in mouse spleen. They were injected i.p. in doses representing approximately 1/8-1/10 of the respective LD50 values. Doses were: DDT, 5.5 mg kg-1 body wt.; malathion, 30 mg kg-1 body wt.; Dursban, 4 mg kg-1 body wt.; Sevin, 7 mg kg-1 body wt.; and Lannate, 1 mg kg-1 body wt. 'Mitomycin C' at a dose of 1 mg kg-1 body wt. was used as a positive control. Mice were sacrificed 6, 24 and 48 h after treatment. DDT, malathion, dursban and lannate caused maximum chromosomal aberrations 24 h after injection, whereas Sevin induced its maximum effect 6 h after the treatment. All the insecticides induced statistically significant chromosomal aberrations even after excluding the number of metaphases with gaps. The results indicate genotoxicity in mouse spleen cells.

Colorimetric determination of cyclophosphamide and ifosphamide.

A simple colorimetric method for the determination of cyclophosphamide and ifosphamide in pure and in dosage forms is suggested. It depends on the reaction of the secondary amino group in both, with nitrous acid, thus forming a nitroso derivative which can be measured at 325 nm for cyclophosphamide and 335 nm for ifosphamide. Beer's law is obeyed with concentrations from 20 to 90 micrograms ml-1 for cyclophosphamide and from 5 to 100 micrograms ml-1 for ifosphadmie, with repeatability of 99.83 +/- 0.256% and 99.72 +/- 0.649%, respectively. Application to different pharmaceutical preparations has shown no significant difference when compared with the B.P. 1988 method.

Toxicological potential of malathion residues in stored soybean seeds.

Succinate-14C-malathion penetrates readily into soybean seeds. The total internal residues inside the seeds amounted to 58-65% of the applied dose after 30 weeks, of which 8-9% were in the form of bound residues. The major part of the internal methanol extractables are chloroform soluble metabolites which include malathion (about 60%), monocarboxylic acid (15%) and its decarboxylation product (8%). The water soluble metabolites contained only one radioactive substance, namely malathion dicarboxylic acid. The toxicological potential of the total internal residues was studied by feeding mice with the washed seeds for about 2.5 months. Treated mice suffered from deterioration of hepatic and renal function as indicated by the observed increased level of blood serum esterases and blood urea nitrogen. The results of blood biochemistry are supported by the histopathological changes observed in the liver, kidney, stomach and intestine. The organs showed degenerative changes including leucocytic aggregation, congestion and dilatation of blood vessels. Other adverse effects caused by malathion residues are indicated from cytogenetic studies on bone marrow of treated mice. Studies showed an initial bone marrow toxicity as indicated by increase in percentage of polychromatic erythrocytes over controls. This effect diminished upon prolongation of feeding period over one month. Feeding with malathion residues affected a gradual increase, with feeding period, in the percentage of polychromatic erythrocytes with micronuclei, a parameter recommended for detecting chemical mutagenes in animal test systems.

Induction of chromosomal aberrations and sister chromatid exchange in vivo and in vitro by the insecticide cypermethrin.

The induction of chromosomal aberrations and sister chromatid exchange in vivo in mouse spleen and bone marrow as well as in vitro in cultured mouse spleen cells by the insecticide 'Cypermethrin' (cis-trans 1:1) was investigated. The percentage of chromosomal aberrations in the spleen and in the bone marrow as almost the same and reached its maximum 6 h following i.p. injection. The aberrations induced were chromatid and chromosome gaps, fragments and tetraploidy. The insecticide caused a significant and dose-dependent increase in the frequency of sister chromatid exchanges (SCEs) in mouse bone-marrow cells: it reached 11.12 +/- 0.05 per cell after treatment with Cypermethrin at 300 mg kg-1 body wt. compared with 3.7 +/- 0.14 per cell and 4.4 +/- 0.26 per cell in the solvent and control, respectively. The percentage of viable cells in mouse spleen cell cultures reached 87.4% and 99.9% relative to the control after treatment of the cell cultures with 10(-3) and 10(-7) Cypermethrin, respectively. All the tested concentrations of Cypermethrin (0.25-400 micrograms ml-1) induced a high percentage of metaphases with chromosomal aberrations after 4 h of treatment. The mean frequency of SCEs per cell reached 15.1 +/- 0.05 after treatment with Cypermethrin at 4.00 micrograms ml-1 compared with 8.6 +/- 0.23 and 5.9 +/- 0.39 in the solvent and control, respectively. The results indicate that Cypermethrin is genotoxic in mouse spleen and bone marrow as well as in cultured mouse spleen cells.

Cytogenetic effects of pesticides. IV. Cytogenetic effects of the insecticides Gardona and Dursban.

The cytogenetic effects of the insecticides Gardona and Dursban were investigated. The toxicity and ability of both insecticides to induce chromosome aberrations and sister-chromatid exchange in vitro was tested in a primary culture of mouse spleen cells, in order to assess the potential mutagenicity of both insecticides. The concentrations 10(-7)-10(-3) M were used for testing the toxic effects of the insecticides. Both Gardona and Dursban were toxic to spleen cell cultures and the percentage of viable cells decreased as the concentration of the insecticide was increased. It reached 76.8% and 77.8% of control after treatment with the highest concentration tested (10(-3) M) of Gardona and Dursban respectively. Gardona at 0.25, 0.50, 1.0 and 2.0 micrograms/ml, and Dursban at 0.50, 1.0, 2.0 and 4.0 micrograms/ml were tested for the induction of chromosome aberrations and sister-chromatid exchanges. All of the tested concentrations of both insecticides induced a high percentage of metaphases with chromosomal aberrations in cultured mouse spleen cells after 4-h treatment. The frequency of SCEs/cell increased with increasing concentration of the insecticides. It reached 11.92 +/- 0.14/cell and 13.40 +/- 0.20/cell after treatment with Gardona (2 micrograms/ml) and Dursban (4 micrograms/ml), respectively, compared with 8.2 +/- 0.19/cell and 7.6 +/- 0.15/cell in the solvent control. The presented results indicate that both Gardona and Dursban in the tested concentrations are mutagenic in mouse spleen cell cultures.

Cytogenetic effects of the insecticide methamidophos in mouse bone marrow and cultured mouse spleen cells.

The cytogenetic effect of the insecticide methamidophos (0,S-dimethylphosphoroamidothiolate) was studied in mouse bone marrow and mouse spleen cells in culture. In vivo the ability of methamidophos to induce micronuclei and sisterchromatid exchange in mouse bone marrow was investigated. In vitro mouse spleen cells in culture were used to assess the ability of the insecticide to induce chromosomal aberrations and sister chromatid exchange. Three different routes of application for the pure insecticide were tested so as to cover the different possibilities for human exposure to the insecticide. Intraperitoneal, oral and dermal treatment with methamidophos caused toxicity to marrow as indicated by a significant increase in the percentage of polychromatic erythrocytes (PEs) over that of the control. Methamidophos showed mutagenic potential as evidenced by a positive response in the micronucleus and chromosomal aberrations assays. Thus, single and multiple i.p. injections at 6 and 4.5 mg methamidophos/kg body wt., oral administration of the insecticide for 14 consecutive days at a dietary level of 100 ppm and multiple dermal treatments (total 4) with 24 mg/kg body wt. induced a statistically significant increase in the frequency of PEs with micronuclei in mouse bone marrow. Moreover, the tested concentrations of methamidophos as low as 0.25 microgram/ml induced a high percentage of metaphases with chromosomal aberrations in cultured mouse spleen cells. Methamidophos is a weak inducer of SCEs in mouse bone marrow and cultured mouse spleen cells.

Cytogenetic effects of pesticides. III. Induction of micronuclei in mouse bone marrow by the insecticides cypermethrin and rotenone.

The production of micronuclei in mouse bone marrow by the pyrethroid insecticide, cypermethrin and the botanical insecticide, rotenone was examined. Three routes of administration were used for the insecticides: intraperitoneal, oral and dermal. The different routes of treatment with cypermethrin and rotenone caused toxicity of marrow as indicated by a significant increase in the percentage of polychromatic erythrocytes (PEs) over that of the control. Cypermethrin showed mutagenic potential as evidenced by a positive response in the micronucleus assay. Oral administration of the insecticide at a dietary level of 900 ppm for 7 and 14 consecutive days as well as double and multiple (total 4) dermal treatments (360 mg/kg body wt.) induced a statistically significant increase in the frequency of PEs with micronuclei. The conducted intraperitoneal (i.p.) treatments with cypermethrin: single injection at 60 and 180 mg/kg body wt., double and multiple injections (total 3) at 60 mg/kg body wt. did not affect the percentage of PEs with micronuclei. The different treatments with rotenone: single, double and multiple (i.p.) injections (total 3) at 2 and 3 mg/kg body wt., oral administration for 14 consecutive days at dietary level of 225 ppm and multiple dermal treatments (total 4) with 135 mg/kg body wt. showed no effect on the frequency of micronuclei in PEs.

Cytological effects of the insecticide tetrachlorvinphos.

The effect of the organophosphorus insecticide tetrachlorvinphos (Gardona) has been studied on the mitosis and meiosis of Vicia faba, using the pure insecticide. An aqueous saturated solution of tetrachlorvinphos affected neither mitosis (after seed-soak and root treatment) nor meiosis (after spraying the plants at the flowering stage). The obtained results may be attributed to the low solubility of tetrachlorvinphos. A saturated solution of tetrachlorvinphos in Tween-60: water mixture (1:99) induced a statistically significant percentage of abnormal cells in root-tip meristems after root treatment for 4 h. Chromosome stickiness, disturbed meta- and anaphases and anaphase bridges were observed.

Cytogenetic effects of pesticides. II. Induction of micronuclei in mouse bone marrow by the insecticide gardona.

The induction of micronuclei in mouse bone marrow by the organophosphorus insecticide gardona (also known as tetrachlorvinphos) was tested. 3 routes of administration were used for the pure insecticide: intraperitoneal, oral and dermal. The different routes of treatment with gardona caused toxicity of marrow indicated as significant increases in the percentage of polychromatic erythrocytes over that of the control. Intraperitoneal and oral treatments induced a statistically significant percentage of micronucleated PE.

Cytogenetic effects of pesticides. I. Induction of micronuclei in mouse bone marrow by the insecticide Dursban.

The induction of micronuclei in mouse bone marrow by the organophosphorous insecticide 'Dursban' was tested. 3 routes of administration were used for the pure insecticide: intraperitoneal, oral and dermal. The different routes of treatment with Dursban induced a statistically significant increase in the percentage of polychromatic erythrocytes over that of the control. Both intraperitoneal and oral treatments with the insecticide induced a high percentage of polychromatic erythrocytes with micronuclei, whereas dermal treatment did not induce micronuclei.

Colorimetric analytical study of oxyphenbutazone.

A procedure was developed for the colorimetric determination of oxyphenbutazone by treatment with dilute nitrite and dilute hydrochloric acid solutions for 10 min, then rendering alkaline with potassium hydroxide. By this simple colorimetric measurement, quantities of oxyphenbutazone from 2.5 to 10 mg/100 ml are determined with an accuracy of 98.80 +/- 1.33%. Application of the suggested method to different pharmaceutical preparations has shown no significant difference compared with the Brit. Ph. 1980 method. A colour reaction mechanism is discussed.

Polarographic analytical study of oxyphenbutazone.

The polarographic behaviour of the widely used anti-inflammatory agent, oxyphenbutazone, was studied. It is determined polarographically by conversion to the nitroso derivative characterized by a cathodic, irreversible, diffusion-controlled wave. The method is applied to the determination of 2.5-10 mg/100 mL of oxyphenbutazone, with an accuracy of 99.9 +/- 1.38%. By differential pulse polarographic analysis, as little as 10 ppm oxyphenbutazone can be determined with an accuracy of 99.70 +/- 0.99% in pure powder and in some pharmaceutical formulations.

Characteristics of chemically transformed mouse epidermal cells in vitro and in vivo.

Primary cultures of epidermal cells from newborn mouse skin have been established. These cultures proliferate and express typical epidermal functions for limited time in vitro. The cultured cells keratinize and exhibit strong reactivity with an epidermis specific membrane antiserum. These cells can be transformed with DMBA either in standard cultures or more easily in cultures using 3T3 feeder cells. Transformed cells have increased proliferation rate. They do not pile up or form multilayered cultures like normal counterparts. They also keratinize less. When transformed cells were grown in the air on collagen gels they formed irregular clumps with central (horn-pearl) keratinization whereas normal controls formed stratified structures. Transofmred cells exhibited reduced reactivity with tissue specific membrane antiserum. There was masking or rearrangement of tissue specific membrane antigens with simultaneous exposition of new fetal-like antigens. Their reactivity with histocompatibility antisera was only slightly reduced. Transformed cells had both numerical and structural chromosome aberrations. They grew in soft agar and they induced tumors upon transplantation in the convenient host. When transplanted as cultures (on collagen) or as suspensions into the host, transformed cells were able to form epithelial cell cords invading the underlying mesenchyme with histological aspect typical of carcinoma. Cell cultures derived from in vivo DMBA induced tumors behaved like in vitro transformed cells.