Induction of CYP1A1, 2B, 2E1 and 3A in rat liver by organochlorine pesticide dicofol.

The present study has determined the ability of dicofol, an organochlorine pesticide, to induce cytochrome P450 using rats treated with 1, 10, and 25mg/kg dicofol intraperitoneally for 4 days. Treatments with 10 and 25mg/kg dicofol produced dose-related increases of cytochrome P450 and cytochrome b(5) contents and NADPH-cytochrome c reductase, 7-ethoxyresorufin O-deethylase, pentoxyresorufin O-dealkylase, aniline hydroxylase, and erythromycin N-demethylase activities in liver microsomes. The treatments also increased glutathione S-transferase and superoxide dismutase activities in liver cytosol. Dicofol at 1mg/kg produced a general trend towards increases of the aforementioned enzyme levels. The results of immunoblot analyses showed that 10 and 25mg/kg dicofol increased protein levels of CYP1A1, CYP2B, CYP2E1, and 3A in liver. RT-PCR data indicated that dicofol induced mRNA expression of liver CYP1A1, CYP2B, and CYP3A. Pretreatments of rats with 10 and 25mg/kg dicofol decreased phenobarbital-induced sleeping time by 34% and 39%, respectively. Dicofol pretreatment at 25mg/kg increased CCl4-induced serum alanine aminotransferase activity by 4.3-fold and aspartate aminotransferase activity by 4.1-fold. The present study demonstrates that dicofol has the ability to induce CYP1A1, CYP2B, CYP2E1, and CYP3A in the liver and increase phenobarbital metabolism and CCl4 toxicity in rats.

Characteristics of 3,5,3'-triiodothyronine (T3)-uptake system of tadpole red blood cells: effect of endocrine-disrupting chemicals on cellular T3 response.

We characterized the 3,5,3'-L-triiodothyronine (T3)- uptake system on the plasma membrane of Rana catesbeiana tadpole red blood cells (RBCs) in the presence of a variety of inhibitors and potentially competing amino acids. Saturable [125I]T3 uptake was inhibited by phloretin, monodansylcadaverine, bromosulfophthalein, sodium taurocholate and tryptophan. Saturable uptake obeyed simple Michaelis-Menten kinetics with an apparent Km of 110 nM and a Vmax of 2.5 pmol/min per 10(6) cells at 23 degrees C. These results suggested that a large proportion of T3 transported into RBCs was mediated by the aromatic amino acid transporter (System T)-linked transporter. To investigate the effect of endocrine-disrupting chemicals (EDCs) on [125I]T3 uptake, RBCs were incubated with [125I]T3 in the presence of each chemical. Among the test chemicals, di-n-butyl phthalate, n-butylbenzyl phthalate and the miticide, dicofol, were the most powerful inhibitors of [125I]T3 uptake, with an IC50 of 2.2 microM, which was one order of magnitude greater than that for T3 (IC50, 0.14 microM), and diethylstilbestrol and ethinylestradiol were modest inhibitors. Tributyltin accelerated saturable initial [125I]T3 uptake by 2-fold at 3.2 microM. When RBCs were cultured with 10 nM T3 at 25 degrees C for 2 days in the presence of monodansylcadaverine, ethinylestradiol, ioxynil or dicofol at the defined concentrations, these compounds inhibited significantly the induction of the thyroid hormone receptor alpha gene by T3. However, not all chemicals competed with T3 binding to the receptor at the same concentrations. Our results raise the possibility that the T3-uptake system on the plasma membrane of the tadpole RBCs could be a candidate target site for some EDCs and can modulate cellular T3 response.

Effects of greenhouse pesticides on the soil-dwelling predatory mite Stratiolaelaps scimitus (Acari: Mesostigmata: Laelapidae) under laboratory conditions.

Knowledge of the effects of pesticides on biological control agents is required for the successful implementation of integrated pest management (IPM) programs in greenhouse production systems. Laboratory assays were conducted to assess the effects of an acaricide (dicofol), two insecticides (chlorpyrifos and pyriproxyfen), and two fungicides (fosetyl-Al and mefenoxam) on Stratiolaelaps scimitus (Womersley), a soil-dwelling predatory mite widely marketed in North America under the name Hypoaspis miles (Berlese) as a biological control agent of dark-winged fungus gnats (Bradysia spp.). Eggs, larvae, protonymphs, deutonymphs, and adult male and female mites were first assayed using dicofol, an acaricide used in the experiments as a positive control, applied to filter paper in an enclosed arena. Protonymphs were assayed for lethal and sublethal effects against the remaining pesticides at maximum label-recommended rates applied to filter paper, by using dicofol as a positive control and water as a negative control. The larva and protonymph were the life stages most susceptible to dicofol, with estimated 24-h LC50 values of 9 and 26 mg m(-2), respectively. Chlorpyrifos was highly toxic to the protonymphs of S. scimitus, causing >95% mortality after 24-h exposure and 100% mortality after 48 h. In contrast, the insect growth regulator (IGR) pyriproxyfen was much less toxic to protonymphs of S. scimitus; pyriproxyfen caused no significant mortality, compared with <5% mortality in the water control. Mortality caused by the fungicides was also relatively low; 72-h exposure to fosetyl-Al and mefenoxam resulted in 17.4 and 27.5% mortality, respectively. The IGR and fungicides increased the duration of the protonymphal stage by 1.2-1.8-fold, but they had no effect on the duration of subsequent life stages, nor on the duration of preoviposition, oviposition, and postoviposition periods of adult females. Total numbers and viability of eggs laid by mites exposed to the IGR and fungicides did not differ from the negative control, although the average rate of egg production during the oviposition of mites exposed to fosetyl-Al was increased. Pyriproxyfen, fosetyl-Al, and mefenoxam are likely to be compatible with S. scimitus under field conditions, because these pesticides caused little mortality of protonymphs, and they did not negatively affect the development and reproduction of S. scimitus under extreme laboratory conditions. In contrast, the use of chlorpyrifos in conjunction with S. scimitus is not recommended unless more comprehensive testing under semifield or field conditions demonstrates compatibility.

Oral estrogen masculinizes female zebra finch song system.

It is well established that parenteral treatment of female zebra finch chicks with estradiol masculinizes their song control nuclei and that as adults they are capable of song. Concern over the widespread use of putative environmental estrogens caused us to ask whether oral exposure to estrogens (a natural route of exposure) could produce similar effects. We dosed chicks orally with estradiol benzoate (EB; 1, 10, 100, and 1000 nmol/g of body mass per day, days 5-11 posthatch), the non-ionic surfactant octylphenol (100 and 1000 nmol/g), or the pesticides methoxychlor (100 and 1000 nmol/g) and dicofol (100 nmol/g) and measured their song control nuclei as adults. EB treatment produced increases in song nuclei comparable to that induced by parenteral administration of estrogens. This is the first study of which we are aware to use an oral route of administration, which simulates the natural process of parent birds feeding their nestlings. We conclude that oral exposure to estradiol alters song control nuclei and we report in a related paper (Millam et al., 2001) that such exposure severely disrupts reproductive performance. Although we detected no influence of xenobiotics on induction of song control nuclei the possibility remains that oral exposure to xenoestrogens in high enough doses could affect development.

Cross-resistance, inheritance, and biochemistry of mitochondrial electron transport inhibitor-acaricide resistance in Tetranychus urticae (Acari: Tetranychidae).

Resistance of the twospotted spider mite, Tetranychus urticae Koch, to acaricides acting as mitochondrial electron transport inhibitors (METIs) is an increasing problem. Because of their high levels of cross-resistance to all commercially available METI-acaricides, a Japanese strain (AKITA) and an English strain (UK-99) of T. urticae were investigated in detail. Larvae of both strains, AKITA and UK-99, showed 1,100- and 480-fold resistance against pyridaben, 870- and 45-fold resistance against fenpyroximate, and 33- and 44-fold resistance against tebufenpyrad, respectively, in a foliar spray application bioassay compared with the susceptible strain GSS. These resistance factors remained stable even when maintained in the laboratory without further selection. Furthermore, strain AKITA showed cross-resistance to dicofol. The METI resistant strains AKITA and UK-99 showed 2.4- and 1.7-fold enhanced O-ethoxycoumarin O-deethylation (cytochrome P450) activity. Increased oxidative metabolism of the METI-acaricides in the resistant strains could be partially suppressed in vivo by the monooxygenase-inhibitor piperonyl butoxide. Reciprocal crosses of homozygous, diploid females and hemizygous, haploid males of strains GSS (susceptible) and AKITA (resistant) revealed that resistance to pyridaben and fenpyroximate was inherited incompletely dominant with slight differences between maternal and paternal inheritance. This is the first attempt to mechanistically describe METI-acaricide resistance in T. urticae. The implications for resistance management strategies are discussed.

Influence of pesticides on counts of Bacillus, heterotrophic bacteria, and fungi in subtropical Brazilian soil microcosms

O crescimento de 200 estirpes de Bacillus, isoladas de solos arenosos, argiloso e orgânico do Rio de Janeiro, foi inibido nas concentraçöes de 10 ppm de Kelthane e 100 ppm de Folidol, testadas com culturas puras. O gênero Bacillus representou uma parcela majoritária da populaçäo de bactérias heterotróficas nesses solos. O efeito dos pesticidas nas contagens microbianas em microcosmos e extrato de solo foi reduzido. Após as primeiras horas de exposiçäo as contagens foram reduzidas em concentraçöes de 100 e 500 ppm, voltando a valores estáveis durante 2 a 4 semanas de experimento. As populaçöes microbianas mistas no solo säo menos afetadas pelos pesticidas que os microorganismos em cultura pura

Two polychlorinated hydrocarbons cause phospholipid-dependent protein kinase C activation in vitro in the absence of calcium.

Polychlorinated hydrocarbons known to be nongenotoxic carcinogens were screened as activators of protein kinase C (PKC)-beta 1 either at high concentrations of Ca2+ or in the absence of Ca2+ (i.e., with 1 mM ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N',-tetraacetic acid). Of those compounds tested, kepone and dicofol significantly stimulated PKC activity in the absence, but not the presence, of Ca2+. PKC activation was most pronounced in the presence of phosphatidylserine. Kepone and dicofol stimulated PKC activity 26% and 13%, respectively, as compared with the PKC activity (100%) stimulated by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Northern blot analysis of expression of TPA-inducible genes by kepone showed slight expression of phorbin and ornithine decarboxylase in murine embryo fibroblasts. Future studies are required to determine the relevance of PKC activation by kepone and dicofol to the known carcinogenicity of these compounds.

Control of polyunsaturated acids in tissue lipids.

Since the discovery in 1929 that certain polyunsaturated fatty acids (PUFA) are essential for life and health, intense investigation has revealed the multiplicity of members in each of several families of PUFA, no two of which are equivalent. The quantified nutrient requirements for the essential dietary precursors of the two dominant families of PUFA have been estimated, and the general functions of these families are slowly becoming known. The PUFA are essential components of structural membrane lipids. The functions of the individual members are not yet differentiated, except as they act as precursors of synthesis of unique octadecanoid, eicosanoid, and docosanoid products of oxidation that have potent biological properties. The PUFA occur in animals and higher plants as ubiquitous and essential components of structural lipid that are in a dynamic equilibrium with the pool of dietary acyl groups. Many human diseases have been found to involve unique essential fatty acid (EFA) deficiencies or distortions of the normal equilibrium pattern. The equilibrium is influenced by the level of dietary intake or precursors, by the presence of competing essential and nonessential acyl groups, by nonoptimum intake of other essential nutrients, by hormonal effects, by drug therapy, and by other effects upon physiological condition. With the many variables already known to modulate or control the equilibrium, it should be possible with more precise understanding of each variable to shift abnormal equilibria in the direction of normalcy. This perhaps will be the next area of intensive investigation in this field of nutrition and metabolism.

Effect of temperature on the inhibition of rat brain synaptosomal ATPases by DDT and its structural analogs.

In vitro effects of DDT and its isomers or metabolites were studied on Na+-K+-ATPase and oligomycin-sensitive (OS) Mg++-ATPase activities at 17, 27 and 37 degrees C in rat brain synaptosomes. Dicofol and methoxychlor were found to be more effective inhibitors of Na+-K+-ATPase and OS Mg++-ATPase than DDT, DDD and DDE. Inhibition of OS Mg++-ATPase by the compounds tested (except DDE) was found to be greater at 17 degrees C than at higher temperatures (17 greater than 27 greater than 37 degrees C), suggesting a negative temperature correlation trend Na+-K+-ATPase was less sensitive to these compounds when compared with OS Mg++-ATPase and the inhibition was greater at higher temperatures (37 greater than 27 greater than 17 degrees C), suggesting a positive temperature correlation trend. Other DDT isomers and metabolites showed variable effects on Na+-K+-ATPase and OS Mg++-ATPases. DDD, but not DDE, inhibited both enzyme activities and the inhibition was independent of temperature. No significant differences were observed in the inhibitory potencies of the various DDT, DDD or DDE ring substitutes studied. The present data indicate that DDT, dicofol and methoxychlor were more effective inhibitors of OS Mg++-ATPase than Na+-K+-ATPase and that the inhibition of the former enzyme had a negative temperature dependence, a feature which parallels toxicity results in insects.

Use of the fluorescent probe, 1-anilino-8-naphthalene sulfonate, to monitor the interaction of pesticide chemicals with mitochondrial membranes.

1. The interaction of mitochondrial and submitochondrial membranes with DDT, methoxychlor and kelthane as monitored by fluorescence changes of ANS was studied. 2. The three organochlorine pesticide chemicals reduced the succinate- and ATP-dependent quenching of ANS fluorescence of mitochondria in a concentration-dependent manner. The results suggest that they blocked the supply of energy to the inner membrane. 3. The reversal of the substrate-induced quenching of ANS by uncouplers was abolished by DDT and kelthane probably as a consequence of the inhibition of protonophoric activity of the uncoupler. 4. Both DDT and kelthane were relatively ineffective inhibitors of substrate-induced quenching of ANS fluorescence of submitochondrial particles. The results suggest an asymmetry of the inner membrane with respect to DDT-inhibition of substrate-induced energization. 5. The possible basis of this asymmetry as well as of inhibition of energy supply to the membrane is discussed.

The cell-wall phosphatase of cotton (Gossypium) is inhibited by kelthane.

Kelthane [4,4'-dichloro-alpha-(trichloromethyl)benzhydrol] was previously shown to decrease the limited tolerance of susceptible varieties of cotton (Gossypium) to Verticillium wilt. Kelthane was shown in the present study to inhibit the cell-wall p-nitrophenyl phosphatase of cotton. In view of information already establishing the cell wall as a primary site of action of Verticillium wilt, the data are interpreted as suggesting an as yet undefined interaction between Kelthane, cell-wall phosphatase and verticillium-resistance mechanisms of the cell wall.

Metabolic and mutagenicity studies on DDT and 15 derivatives. Detection of 1,1-bis(p-chlorophenyl)-2,2-dichloroethane and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethyl acetate (kelthane acetate) as mutagens in Salmonella typhimurium and of 1,1-bis(p-chlorophenyl) ethylene oxide, a likely metabolite, as an alkylating agent.

Using a novel in vitro technique, whereby microsomal enzymes were embedded in an agar layer to prolong their viability, 1,1-bis(p-chlorophenyl) ethylene(DDNU), a mammalian metabolite of 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), was converted by microsomal mono-oxygenases of mouse liver into 1,1-bis(p-chlorophenyl)-1,2-ethanediol (DDNU-diol). The putative epoxide intermediate, 1,1-bis(p-chlorophenyl)ethylene oxide (DDNU-oxide), a new compound, was synthesized; it showed weak alkylating activity with 4-(4-nitrobenzyl)pyridine but was not mutagenic in Salmonella typhimurium strains TA100 and TA98. DDT and 13 of its metabolites or putative synthetic derivatives, including 1,1-bis(p-chlorophenyl)-2,2-dichloroethylene (DDE), 1 1,1-bis(p-chlorophenyl)-2-chloroethylene (DDMU), 1,1-bis(p-chlorophenyl)-2-chloroethane (DDMS)-DDNU, 2,2-bis(p-chlorophenyl)ethanol (DDOH), bis(p-chlorophenyl)acetic acid (DDA) and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethanol (Kethane), caused no mutagenic effects in S. typhimurium strains TA100 or TA98, either in the presence or absence of a mouse-liver microsomal fraction. 1,1-Bis(p-chlorophenyl)-2,2,2-trichloroethyl acetate (Kelthane acetate) was a direct-acting mutagen in strain TA100, whereas 1,1-bis(p-chlorophenyl)-2,2-dichloroethane (DDD) was mutagenic in TA98, only in the presence of a mouse-liver microsomal system. The results are discussed in relation to possible pathways whereby DDT is activated to mutagenic and/or carcinogenic metabolites.

Residue analysis of DDT, malathion and kelthane on pears.

Malthion, DDT, kelthane and their mixture (3 : 10 : 5) were applied to pear fruits at the rate recommended by the Ministry of Agriculture. The rate of disappearance of their residues on pear fruits was investigated under refrigerated conditions (2 +/- 1 degree and 47% relative humidity (RH)). The same insecticides were tested in higher concentrations (1.5 and 2 times) to determine the effect of insecticides on the black spots usually found on pears after storage. No relation was found between the tested insecticides and the appearance of the black spots. Residue-disappearance curves of the three tested concentrations of these insecticides and their mixture were drawn on semi-log paper. The residues of each material found on pear fruits after one hour, 1, 8, 15, 30 and 45 days' storage in a refrigerator, estimated biologically by C. pipiens larvae and D. magna were recorded. The highest concentrations of the mixture and of the individual insecticides left higher residues on pears than the lower ones of all the insecticides either as a mixture or alone. Stored pears, refrigerated under the above conditions immediately after treatment with the mixture of insecticides recommended for the pest control of pears, cannot be consumed fresh after 45 days.