Role of cytochrome P450s in insecticide resistance: impact on the control of mosquito-borne diseases and use of insecticides on Earth.

The fight against diseases spread by mosquitoes and other insects has enormous environmental, economic and social consequences. Chemical insecticides remain the first line of defence but the control of diseases, especially malaria and dengue fever, is being increasingly undermined by insecticide resistance. Mosquitoes have a large repertoire of P450s (over 100 genes). By pinpointing the key enzymes associated with insecticide resistance we can begin to develop new tools to aid the implementation of control interventions and reduce their environmental impact on Earth. Recent technological advances are helping us to build a functional profile of the P450 determinants of insecticide metabolic resistance in mosquitoes. Alongside, the cross-responses of mosquito P450s to insecticides and pollutants are also being investigated. Such research will provide the means to produce diagnostic tools for early detection of P450s linked to resistance. It will also enable the design of new insecticides with optimized efficacy in different environments.

Ultrastructural effects of DDT, DDD, and DDE on neural cells of the chicken embryo model.

Human exposure to environmental compounds with estrogenic activity and the potential effects on human health is the subject of ongoing scientific debates. Their potential effects raise concern regarding neurological development after prenatal exposure. Central to this debate is the pesticide 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT). Although it has apparent low acute toxicity in mammals, DDT has a long residual persistence and laboratory research has indicated that it acts on the CNS by interfering with Na(+)/K(+) pump mechanism of the neuronal membranes, causing disruption in Ca(2+) homeostasis. Potentially this may lead to both apoptosis and necrosis. The present study investigates the effects of DDT and two of its metabolites DDD and DDE on the ultramorphology of neural cells, using a previously published chicken embryo model. Results indicate cellular swelling, budding, and increased membrane permeability for all three chemicals, accompanied by karyolysis in the DDE group (typical features of oncosis). These results support the finding of other researchers as well as the concerns of the WHO that DDT and its metabolites may cause neurotoxicity after prenatal exposure.

DDT and its metabolites alter gene expression in human uterine cell lines through estrogen receptor-independent mechanisms.

Endocrine-disrupting organochlorines, such as the pesticide dichlorodiphenyltrichloroethane (DDT), bind to and activate estrogen receptors (ERs), thereby eliciting estrogen-like effects. Although ERs function predominantly through activation of transcription via estrogen-responsive elements, both ERs, alpha and ss, can interact with various transcription factors such as activator protein-1 (AP-1). Additionally, estrogens may regulate early signaling events, suggesting that the biological effects of environmental estrogens may not be mediated through classic ER (alpha and ss) activity alone. We hypothesized that known environmental estrogens, such as DDT and its metabolites, activate AP-1-mediated gene transactivation through both ER-dependent and ER-independent means. Using two Ishikawa human endometrial adenocarcinoma cell line variants that we confirmed to be estrogen responsive [Ishikawa(+)] and estrogen unresponsive [Ishikawa(-)], we generated stably transfected AP-1 luciferase cell lines to identify the role of an estrogen-responsive mechanism in AP-1-mediated gene expression by various stimuli. Our results demonstrate that DDT and dichlorodiphenyldichloroethane (DDD) were the most potent activators of AP-1 activity; 2,2-bis(p-chlorophenyl) acetic acid failed to activate. Although stimulated in both Ishikawa(+) and Ishikawa(-) cells by DDT and its congeners, AP-1 activation was more pronounced in the estrogen-unresponsive Ishikawa(-) cells. In addition, DDT, DDD, and dichlorodiphenyldichloroethylene (DDE) could also stimulate AP-1 activity in the estrogen-unresponsive human embryonic kidney 293 cells using a different promoter context. Thus, our data demonstrate that DDT and its metabolites activate the AP-1 transcription factor independent of ER (alpha or ss) status.

Six high-priority organochlorine pesticides, either singly or in combination, are nonestrogenic in transfected HeLa cells.

There have been increasing concerns that environmental chemicals may adversely affect the health of humans and wildlife by acting as endocrine modulators. These concerns have been augmented by the realization that human exposure occurs not just to single chemical agents, but typically to mixtures of chemicals that could exhibit estrogenic activity qualitatively and/or quantitatively different from that of individual components. To address these concerns, we have evaluated the ability of six organochlorine pesticides (4, 4'-DDT, 4,4'-DDD, 4,4'-DDE, aldrin, dieldrin, or endrin, all classified high priority by ATSDR) to modulate transcriptional activation of an estrogen-responsive reporter gene in transfected HeLa cells. In these assays, HeLa cells cotransfected with an expression vector encoding estrogen receptor and an estrogen-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid were exposed to these pesticides individually and in defined combinations. While estradiol consistently elicited 10- to 23-fold dose-dependent inductions in these assays, the six organochlorine pesticides showed no detectable dose-related response when tested individually. When tested in binary combinations, the pesticide mixtures showed no additional estrogenicity. Thus, the pesticides tested, singly or as mixtures, showed virtually no evidence of estrogenicity.

Comparative pharmacodynamics of CYP2B induction by DDT, DDE, and DDD in male rat liver and cultured rat hepatocytes.

In this study the pharmacodynamics were characterized of rat hepatic cytochrome P-450 2B (CYP2B) induction by the pesticide DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolites DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], which is bioretained, and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], which is metabolized further and therefore less prone to bioaccumulate. DDT, DDE, and DDD were each found to be pure phenobarbital-type cytochrome P-450 inducers in the male F344/NCr rat, causing induction of hepatic CYP2B and CYP3A, but not CYP1A. The ED50 values for CYP2B induction (benzyloxyresorufin O-dealkylation) by DDT, DDE, and DDD were, respectively, 103, 88, and > or = 620 ppm in diet (14 d of exposure). The efficacies (Emax values) for induction of benzyloxyresorufin O-dealkylation by DDT, DDE, and DDD were 24-, 22-, and > or = 1-fold, respectively, compared to control values. The potencies of the three congeners for CYP2B induction appeared also to be similar, with EC50 values (based on total serum DDT equivalents) of 1.5, 1.8, and > or = 0.51 microM, respectively. The EC50 values based on DDT equivalents in hepatic tissue were 15, 16, and > or = 5.9 micromol/kg liver tissue, respectively. In primary cultures of adult rat hepatocytes, DDT, DDE, and DDD each displayed ability to induce total cellular RNA coding for CYP2B (ED50 values of 0.98, 0.83, and > or = 2.7 microM, respectively). These results suggest that DDT, DDE, and DDD each possess a high degree of intrinsic CYP2B-inducing ability for rat liver, despite marked differences in bioretention among the congeners.

Effects of perinatal polychlorinated biphenyls and dichlorodiphenyl dichloroethene on later development.

OBJECTIVE: Determining whether early developmental effects of perinatal exposure to polychlorinated biphenyls (PCBs) or dichlorodiphenyl dichloroethene (DDE) persist. DESIGN: Cohort followed from birth; ages now 5 1/2 to 10 1/2 years. SETTING: General community. PARTICIPANTS: Volunteer sample of 859 children, of whom 712 had been examined with the McCarthy Scales of Children's Abilities at 3, 4, or 5 years; 506 sent report cards. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: Neither transplacental nor breast-feeding exposure to PCBs or DDE affected McCarthy scores at 3, 4, or 5 years. There was no statistically significant relationship between poorer grades and PCB or DDE exposure by either route. CONCLUSIONS: The deficits seen in these children on the Bayley Scales of Infant Development through 2 years of age are no longer apparent.

A study of the effects of p,p'-DDE and other related chlorinated hydrocarbons on inhibition of platelet aggregation.

A series of chlorinated hydrocarbons of interest in environmental toxicology were investigated concerning their effects on human platelet aggregation. Most potent in inhibiting platelet aggregation were p,p'-DDE and Arochlor 1242. Aggregation induced by arachidonic acid (1 mM) was more sensitive to inhibition by p,p'-DDE than was aggregation induced by ADP (10 microM). The former was completely inhibited by p,p'-DDE at a concentration of 1 x 10(-4) M, whereas there was only a 31% inhibition of the latter. Addition of Ca2+ (1 mM) blocked the inhibitory effect of p,p'-DDE on aggregation induced by both arachidonic acid and ADP. Calmodulin (1 microgram/ml) blocked the inhibitory effect of p,p'-DDE on aggregation induced by arachidonic acid but not that induced by ADP. The calmodulin inhibitory drugs trifluoperazine and calmidazolium had no effect at all or only a weak effect (-14%), respectively, on platelet aggregation. Increasing the concentrations of p,p'-DDE and Arochlor 1242 caused a delay in the onset of aggregation induced by the addition of arachidonic acid. The synthesis of thromboxane B2 and other prostaglandins in platelet membranes was dose-dependently reduced by p,p'-DDE. The structurally closely related isomers o,p'-DDE and p,p'-DDT did not significantly inhibit arachidonic acid-induced platelet aggregation or thromboxane B2 synthesis. It is concluded that p,p'-DDE and Arochlor 1242 inhibited platelet aggregation by inhibiting platelet cyclooxygenase activity.

Effects of fasting, water deprivation, and adrenal-blocking chemicals on resistance to Escherichia coli challenge.

Fasting for 2 days plus 1 day of feeding resulted in increased resistance to Escherichia coli challenge. If feed during the postfasting period contained metyrapone (a blocker of adrenal synthesis of corticosterone), resistance to E. coli challenge was reduced. Fasting for 1.75 days or water deprivation for 2 days resulted in decreased resistance to E. coli challenge. Deprivation of water for two days of chickens with blocked adrenals resulted in an E. coli challenge response similar to that of controls.

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.

Evaluation of the activity of chlorine-containing pesticides as inhibitors of the mitochondrial respiratory chain.

The influence of chlorine-containing pesticides, which are representatives of various classes of chemical compounds and possess various pesticidal activities, on the NAD . H oxidase activity of the submitochondrial particles (SMP) obtained from beef heart was studied. It was found that the strongest inhibitors of NAD . H oxidase of the SMP are chloro-derivatives of aromatic hydrocarbons (DDT and its analogs DDD and DDE). Derivatives of propionic and acetic acids (propinate, dalapon, MCAA, DCAA, and TCAA) did not exhibit any appreciable inhibiting effect under the experimental conditions. Criteria were introduced (phi and Ca50) permitting a quantitative characterization of the influence of pesticides on the respiratory chain of the mitochondria. Indications were obtained of possible mechanisms of the inhibiting action of the pesticides used.

Pesticide-induced changes in hepatic microsomal enzyme systems: further studies on the effects of 1,1,-di(p-chlorophenyl)-2-chloroethylene (DDMU) in the Japanese quail.

Changes in the liver resulting from the low level dietary administration of 1,1-di(p-chlorophenyl)-2-chloroethylene (DDMU),p,p'-DDT, o,p'-DDT, p,p'-DDD and p,p'-DDE to Japanese Quail have been monitored. DDMU was exceptional in causing substantial increases in relative liver wt. and hepatic glucose-6-phosphatase after feeding at 100 ppm for 28 days. The time course of liver enzyme induction by DDMU has also been studied in Japanese Quail after periods of dietary administration ranging from 1--28 days with particular reference to changes in hepatic cytochrome P-450 and relative liver wt. Structural changes in the liver have been followed by reference to protein and lipid components. The hepatic response to DDMU appears to be biphasic. Initially there are substantial increases in hepatic cytochrome P-450 and relative liver wt., but the latter is largely due to accumulation of triglycerides. After approximately 20 days the level of hepatic cytochrome P-450 remain at a high 'plateau' level. This secondary phase of liver induction probably involves cell proliferation. It is concluded that DDMU causes major changes in the avian liver and either directly or through a metabolite causes pronounced microsomal enzyme induction.

[Action of O,N'-dichlorodiphenyldichloroethane (O,N'-DDD) on adrenal cortical ATPase]. / Vozdeistvie O,N'-dikhlordifenildikhlorétana (O,N'-DDD) na ATF-azy kory nadpochechnikov.

The action of the inhibitor of the adrenal gland function of dogs--o,n'-DDD--on ATP-ases was studied experimentally. In a concentration of 0.2--1.0 mM o,n'-DDD inhibited the Na+, K+. Mg2+-ATP-ases of the homogenate, and also the mitochondrial and microsomal fractions of the dog adrenal cortex. The effect of the inhibitor became intensified with the temperature elevation. An inhibition of ATP-ases in the homogenate and the microsomal fraction was noted as a result of a single feeding of o,n'-DDD to dogs (50 mg per 1 kg of body weight). A double o,n'-DDD feeding caused activation of the enzymes in the homogenate and the mitochondrial fraction.

[Modifying effect of DDT and of its various metabolites on the toxic effect of 7,12-dimethylbenz(alpha)anthracene]. / Modifitsiruiushchee deistvie DDT i nekotorykh ego metabolitov na toksicheskii effekt 7,12-dimetilbenz(alpha)antratsena.

In rats a preliminary three times injection of DDT in doses of 10 and 100 mg/Kg eliminated completely DMBA (30 mg/per 100 g of weight) lethal effect and prevented or decreased the development of adrenal necrosis. Among the metabolites under study (DDT, DDON, DDMU, DDE, DDA, DVR) DDE was found to render the strongest protective action in its single administration in a dose of 10 mg/Kg a day before the exposure to DMBA.

Interactions of chlorinated hydrocarbons with steroid hormones.

Effects of chlorinated hydrocarbons of the DDT class in mammals were examined. Two effects were considered: a) induction of hepatic mixed function oxidase (MFO), and b) estrogenic activities. The induction of MFO was discussed primarily with reference to the enhancement of steroid hydroxylation. In turn, the increase in steroid hydroxylation by chlorinated hydrocarbons was usually accompanied by a decrease in the biological activity of endogenous and administered steroid hormones. The estrogenic activity of chlorinated hydrocarbons was found to reside primarily in the o,p'-DDT (a major contaminant of technical grade DDT). The mechanism of the estrogenic activity by DDT homologs was explored. It appears that o,p'-DDT acts like estradiol (E2). Similarly to E2, o,p'-DDT binds to the uterine cytosolic receptor. Furthermore, like E2, o,p'-DDT is a potent inducer of certain uterine enzymes. For instance induction of ornithine decarboxylase of about 200-fold was observed with a high dose (250 mg/kg body wt); however as little as 5 mg/kg of o,p'-DDT exhibited marked induction of this enzyme--about threefold. The above activities of the chlorinated hydrocarbons were considered with respect to the potential long-term toxic effects which these compounds might elicit.

[Effect of adrenocortical insufficiency on the motor activity of the gall bladder]. / Vliianie nedostatochnosti funktsii kory nadpochechnikov na motornuiu deiatel'nost' zhelchnogo puzyria

In chronical experiments on dogs, blocking of glococorticoidal function of adrenal cortex O, pI -- DDD decreases the gall bladder motility. Disorders of mineralcorticoid activity of adrenal cortex leads to an increase of the motility. Administration of hydrocortisone and 1% solution of sodium chloride aids to normalize the gall bladder motility in animals with adrenal insufficiency.

The effect of insecticides on Chinese hamster cell cultures.

The effect of p,p'-isomers of DDT and its derivatives DDD, DDE and DDA on Chinese hamster cells in culture was studied. At different concentrations and various times of treatment the proliferation rate was inhibited most strongly by DDD and DDT, whereas DDE exhibited a markedly weaker influence. DDA was the least toxic compound of the four. The cytogenetic effects were also different. Again, DDA induced the least damage. Only enhanced gap rates but no chromosome breaks were observed. DDE was more active, and higher break rates occurred. DDD and DDT were by far the most damaging compounds, and they raised the gap and break rates markedly. However, no induction of configuration anomalies was found in any experiment. Chronic treatment of the cells for 3 months with DDT at 8 ppm did not alter the proliferation rate, the sensitivity to acute treatment with higher DDT concentrations or the chromosomal aberration rates. The results are discussed in relation to the relevance of differential pesticide effectivity in organs of higher animals and man.