Studies on the induction of rat uterine ornithine decarboxylase by DDT analogs. I. Comparison with estradiol-17β activity

The effect of DDT analogs and estradiol-17β on uterine ornithine decarboxylase activity in the immature intact and ovariectomized rat was studied. Pretreatment with various doses of o,p′DDT [1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane] or estradiol-17β caused a marked increase in the specific activity of ornithine decarboxylase in the 20,000g supernatant fraction of uterine homogenates but not in liver homogenates. Doses as low as 0.5 mg of o,p′DDT or 0.002 μg of estradiol-17β stimulated uterine ornithine decarboxylase activity in the ovariectomized rat. The peaks of activity after treatment with o,p′ DDT and estradiol-17β occurred at 6 and 5 hr, respectively. The level of ornithine decarboxylase activity in untreated groups was consistently lower in ovariectomized rats than in intact immature animals. Treatment with o,p′ DDT (10 mg/100 g body weight) of ovariectomized and intact immature rats demonstrated at 131-fold and an about 20-fold increase in uterine ornithine decarboxylase activity, respectively. Treatment of ovariectomized rats with cycloheximide or actinomycin D effectively blocked the increase in ornithine decarboxylase caused by o,p′ DDT. Similar results were obtained with cycloheximide in the intact immature rat. Animals subjected to both adrenalectomy and ovariectomy demonstrated an increase in ornithine decarboxylase activity when treated with either estradiol-17β or o,p′ DDT. Dose-response curves obtained for estradiol-17β and o,p′ DDT suggest a similar mechanism of action for the two compounds. Graphic analysis of the dose-response curves for estradiol-17β and o,p′ DDT demonstrated an ED₅₀ of 0.038 μg/100 g body weight and 1.8 mg/100 g body weight, respectively. The examination of various DDT analogs in intact and ovariectomized animals showed that o,p′ DDT was the most potent inducer of ornithine decarboxylase. The order of decreasing potency of DDT analogs was o,p′ DDT, o,p′ DDD. p,p′ DDT, p,p′ DDD, and p,p′ DDE.     

The metabolism of p,p′-DDT by the feral pigeon (Columba livia)

Male feral pigeons were dosed with ring-labeled $\text{[}{}^{14}\text{C}\text{]p,p′-}\text{DDT}$ and the tissues and droppings analyzed for total ¹⁴C, extractable ¹⁴C, and metabolites. Only 16% of an intraperitoneal dose of 1.5–2.2 mg kg^?1 was voided in the droppings over 28 days; the rate of loss reached a maximum on the 14th day and then fell quickly away. The rate of removal of ¹⁴C in droppings was low in comparison to that found in the rat and the Japanese quail. When pigeons were dosed with 32–38 mg kg^?1 DDT per bird, and killed after 77 days, 5.4% of the dose was eliminated in droppings and 87% was recovered in the body. The tissues and droppings from this experiment were analyzed for DDT and its metabolites. Of the ¹⁴C remaining in tissues 88% was accounted for as the apolar compounds DDE, DDT, and DDD. Approximately half of the ¹⁴C in droppings was present as DDE, DDT, and DDD, whereas 27–35% was apparently in conjugated form, extractable from aqueous solutions by ethyl acetate after prolonged acid hydrolysis. Two polar metabolites were isolated from the acid-released material. One was p,p′-DDA; the other was extractable from aqueous solution at pH 8 and was tentatively identified as a monohydroxy derivative of p,p′-DDT. DDE accounted for 93% of the ¹⁴C present as metabolites in tissues and droppings, clearly indicating the importance of this intermediate in this study. The metabolism of DDT in the feral pigeon is discussed in relation to its metabolism by other species.     

Metabolism of methylated analogs of aldrin and dieldrin by liver preparations from male and female rat

Analogs of aldrin and dieldrin have been synthesized, the molecules of which lack a methylene bridge but have two methyl groups attached to olefinic or oxirane ring carbon atoms. Their metabolism was studied using NADPH-supplemented 12,000g × 30-min supernate of livers from Wistar and CD strains of rats. The dimethylated olefin was not metabolized to its epoxide but was converted by supernate of liver from rats of both sexes to a labile hydroxylated metabolite. Exogenous NADPH increased the rate at which the olefin was metabolized, but concentrations of NADPH which increased the yield of the labile metabolite when added to female rat liver preparations decreased the yield when added to those from the male. This metabolite had a mass spectrum which was consistent with it being a hydroxymethyl derivative. The dimethylated epoxide was metabolized more slowly than the olefin, and a notable sex difference in the route of metabolism was evident. With supernate from livers of female rats the only metabolite detected by gas-liquid chromatography had a mass spectrum consistent with it being a ring-hydroxylated epoxide. Male rat liver supernate formed small quantities of the same compound, but the major metabolite had a mass spectrum indicative of a hydroxymethyl epoxide. No hydrolysis of the oxirane ring of the epoxide has been detected. The relevance of these results to the planned synthesis of cyclodienes of optimal biodegradability is mentioned.     

Anaerobic metabolism of DDT analogs by pigeon liver preparations

The amounts of p,p′-DDT and of five other trichloroethane derivatives decreased upon incubation under anaerobic conditions with 12,000g × 30 min pigeon liver supernatant fraction. The addition of an exogenous NADPH-generating system sometimes, but not invariably, increased the rate of metabolism. Only one hexane-soluble metabolite was detected in the postincubation reaction medium of each of the six trichloroethane derivatives. After isolation by tlc the six metabolites were shown by mass spectrometry to have molecular weights 34 units less than their parent compounds. Comparison of the isotope patterns in the spectra of each substrate and its metabolite reveals that in each case the metabolism involves the loss of a chlorine atom. From these data it is concluded that several substituted trichloroethanes undergo reductive dechlorination when they are incubated with liver preparations in an atomosphere of nitrogen. Two dichloroethane derivatives, tested in a similar manner, were unchanged and were recovered quantitatively. Mass spectrometric and chromatographic data of reactants and products are recorded.     

Factors involved in increased protein synthesis in liver microsomes after administration of DDT

Microsome fractions were prepared from liver homogenates of control rats and rats treated with DDT. The increased incorporation of [¹⁴C]phenylalanyl-tRNA into peptide when microsomes or ribosomes derived from DDT-treated rats were incubated with supernatant was due to factors in addition to increased endogenous mRNA. These factors were not related to the decreased activity of ribonuclease and increased activity of ribonuclease inhibitor, nor to the GTP and thiol sensitive inhibitor. The factors were partly removed from microsomes and completely removed from ribosomes by procedures using a 0.5 M KCl wash. Treatment of rats with polychlorinated biphenyls gave results similar to DDT. The 0.5 M KCl wash fraction from the control preparations contained factors inhibitory to protein synthesis in contrast to the wash fraction from the DDT preparations.     

The postmortem reductive dechlorination of pp′DDT in avian tissues

Pigeons, Bengalese finches, and Japanese quail were dosed with pure pp′DDT, and their tissues were examined for residues immediately after death, or following storage. DDD residues in livers extracted immediately after death were always low (<5% DDT concentration) and sometimes undetectable. Reductive dechlorination of DDT to DDD occurred postmortem, relatively repidly at 20°C (90% after 8 hr), but more slowly at ?10 and ?20°C. The conversion could not be attributed to bacterial action and was evidently due to processes operating within the cell under anaerobic conditions. During storage at ?12 to ?15°C, reductive dechlorination occurred in brain and in embryos, but did not occur to any significant extent in depot fat or in eggs without embryos. These findings raise questions about the importance of DDD as an in vivo metabolite in birds and other vertebrates. The interpretation of DDD residues in tissues from laboratory and field studies is discussed.     

Components of the electron transport system in the microsomal mixed-function oxidase system in wild birds

Notable differences were found among six species of wild-caught birds in the levels of cytochrome P-450, cytochrome b₅, NADPH-cytochrome c reductase, and NADH-cytochrome c reductase. Ethyl isocyanide difference spectra showed significant variations among the species in peak height and in the ratios of the $\text{430}\text{455}\text{-}\text{nm}$ peaks. Substantial aldrin epoxidase activity was found in all species, and the amounts of dieldrin produced compared favorably with pigeon and rat liver microsomes. Higher content of cytochrome P-450 was not always accompanied by a similar rise in specific catalytic activity. Thus, no correlation could be established between these two parameters. Aldrin epoxidase activity with NADH as the sole electron donor was 25–49% as effective as with the NADPH-generating system. Addition of both NADH and NADPH-generating systems to the incubation mixture produced a synergistic effect with liver microsomes of two species but not with two other species. DDE and polychlorinated biphenyls residues were found in the heart tissue of all species examined, and this might indicate a possible inductive effect on the microsomal mixed-function oxidase system by environmental contaminants.     

In vitro metabolism of EPN and EPNO by mouse liver

The in vitro metabolism of EPN (O-ethyl O-p-nitrophenyl phenylphosphonothionate) and EPNO (O-ethyl O-p-nitrophenyl phenylphosphonate) in mouse liver was studied. EPNO was metabolized faster than EPN, and the highest metabolic activity was found in the 10,000g supernatant in the presence of both NADPH and glutathione. Liver microsomes in the presence of NADPH metabolize EPN to its oxygen analog, EPNO and p-nitrophenol. With the 100,000g supernatant only slight metabolism of EPN occurred in the presence of GSH. Metabolism of EPNO by liver microsomes increased upon the addition of NADPH. p-Nitrophenol was the only metabolite isolated in the presence of microsomes, whereas, with the addition of NADPH, both p-nitrophenol and desethyl EPNO were formed. Quantitative studies showed that there was little, if any, oxidative dearylation of EPNO by liver microsomes. The 100,000g supernatant was found to actively degrade EPNO, and this increased upon addition of glutathione. The initial rate of p-nitrophenol formation as a result of incubation of EPN and EPNO with liver microsomes was found to be higher with EPN than EPNO.     

Subcellular distribution of malathion,phenthoate, and diethylsuccinate carboxylesterases in rat lungs

The subcellular distribution of malathion, phenthoate, and diethylsuccinate carboxylesterases was determined in the lungs of male Sprague-Dawley rats and in rats administered lung toxic doses of bromobenzene and paraquat. In control with the former two substrates, the highest activity was encountered in the 6500g and 12,000g pellets. In addition, significant activity was found in the 100,000g supernatant. These fractions hydrolyzed diethylsuccinate very slowly, and the major diethylsuccinate carboxylesterase activity was recovered in the 100,000g pellet. The bromobenzene treatment had no effect on these carboxylesterases; however, the paraquat treatment significantly decreased the phenthoate and diethylsuccinate carboxylesterases in the 100,000g pellet without altering the activity in the other fractions. The present study suggested that the subcellular distribution of malathion and phenthoate carboxylesterases is different from that in liver. The present study revealed that, in the lungs, the highest total activity for malathion and phenthoate carboxylesterases was found in the 100,000g supernatant, in comparison with liver, where the 100,000g pellet contains a much higher activity of these enzymes. The decrease in specific activities of diethylsuccinate and phenthoate carboxylesterases following the treatment with a pneumotoxicant may serve as an indicator of lung damage.     

The metabolism of DDT in vivo by the Japanese quail (Coturnix coturnix japonica)

Male and female Japanese quail (Coturnix coturnix japonica) were given intraperitoneal injections of [¹⁴C]DDT in ethanol at a rate of 13.4 mg/kg body wt. Fifty-six days later the tissues and droppings were analysed for total ¹⁴C and metabolites. The rate of loss of ¹⁴C in droppings was very similar in males and females. The maximal rate was reached on the third day, and 65–66% of the injected dose was voided by the fifty-sixth day. Ninety-three to ninety-four percent of the ¹⁴C in droppings and 83–90% of the ¹⁴C in tissues were extracted by solvents. Combined extracts from males and females were used for determination of DDT and its metabolites. Expressing all results as percentages of injected dose, the following were isolated from droppings: DDA (24%), DDT (3%), DDD (5.1%), DDE (11%), and uncharacterised polar metabolites (17%). Twenty-five percent of the dose was retained in the tissues and this was largely accounted for as DDT (10.4%) and DDE (10.5%). Of the total metabolites found 31% was DDE (almost equally divided between tissues and droppings) and 35% was DDA (almost entirely in droppings). Since DDD was not found in significant quantities in tissues, the substantial quantities in droppings were probably produced from DDT by the action of microorganisms.     

Resistance in the highly DDT-resistant 91-R strain of Drosophila melanogaster involves decreased penetration,increased metabolism,and direct excretion

Resistance to 4,4′-dichlorodiphenyltrichloroethane (DDT) in the 91-R strain of Drosophila melanogaster is extremely high compared to the susceptible Canton-S strain (>1500 times). In addition to enhanced oxidative detoxification, the 91-R strain also has a reduced rate of DDT penetration, increased levels of reductive and conjugative metabolism, and substantially more excretion than the Canton-S strain. Contact penetration of DDT was ∼30% less with 91-R flies, which also had significantly more cuticular hydrocarbons and a thicker, more laminated cuticle compared to Canton-S flies, possibly resulting in penetration differences. DDT was metabolized ∼1.6-fold more extensively by 91-R than Canton-S flies, resulting in dichlorodiphenyldichloroethane (DDD), two unidentified metabolites and polar conjugates being formed in significantly greater amounts. 91-R flies also excreted ∼4-fold more DDT and metabolites than Canton-S flies. Verapamil pretreatment reduced the LD₅₀ value for 91-R flies topically dosed with DDT by a factor of 10-fold, indicating that the increased excretion may involve, in part, ATP-binding cassette (ABC) transporters. In summary, DDT resistance in 91-R is polyfactorial and includes reduced penetration, increased detoxification and direct excretion.     

Uptake,metabolism and effects of DDT,fenitrothion and chlorpyrifos on Tetrahymena pyriformis

The uptake and metabolism of DDT, fenitrothion and chlorpyrifos were studied in cultures of the ciliate protozoan Tetrahymena pyriformis. When cultures were treated with DDT in concentrations varying from 0.01 to 0.5 μg ml⁻¹, concentrations found in T. pyriformis were 3.8 to 335 μg g⁻¹ dry weight. The accumulation of fenitrothion ranged from 28.7 μg g⁻¹ in cultures treated with 1 μg ml⁻¹ to 2260 μg g⁻¹ in cultures treated with 10 μg ml⁻¹. Under similar experimental conditions chlorpyrifos was accumulated from 24.7 to 15400 μg g⁻¹. The patterns of uptake were dependent on the growth cycle, the ability of the organism to metabolise insecticide and the type of the insecticide used. Maximum accumulation of DDT, fenitrothion and chlorpyrifos occurred in 2, 4 and 6 h respectively. Tetrahymena metabolised DDT to DDD and DDE but failed to metabolise fenitrothion and chlorpyrifos. The effects on growth and morphology of T. pyriformis were studied over a period of 5 days. Higher concentrations (10, 50 and 100 μg ml⁻¹) of DDT inhibited only the growth of the organisms and did not change cell morphology. Fenitrothion was extremely toxic to the organisms and at 5 and 10 μg ml⁻¹ cells became more or less spherical and died after 48 h. However, concentrations of 0.5, 1 and 2.5 μg ml⁻¹ fenitrothion caused growth inhibition, but only at 2.5 μg ml⁻¹ was this permanent. Chlorpyrifos inhibited the growth of the organisms at 1, 5 and 10 μg ml⁻¹ but the morphology was affected only at 5 and 10 μg ml⁻¹.     

Insecticidal and neuroexciting actions of DDT analogs

The neuroexciting activity of DDT and its analogs to produce repetitive responses on the nerve cord of Periplaneta americana was determined using the extracellular electrode method. The convulsive activity on P. americana and the insecticidal effect on Callosobruchus chinensis were also examined. It was found that the convulsive and insecticidal activities increase almost proportionally with increase in the neuroexciting activity within a set of p,p′-substituted DDT analogs. The intimate connections among these biological effects suggest that symptoms such as convulsion and death caused by DDT analogs are closely related with their neuroexcitory effect and there is a common mode of action in spite of differences in insect species.     

Comparative enzyme activities and cytochrome P-450 levels of some rat tissues with respect to their metabolism of several pesticides

Cytochrome P-450, A- and B-esterase, amidase, and glutathione S-aryl transferase were assayed in the postmitochondrial centrifugal fraction, microsomes, and supernatant of rat liver, lungs, kidneys, and testes. Liver microsomes contained the highest P-450 levels and A-esterase activity. B-esterase activity was more generally distributed and higher in the microsomal tissue fractions. Microsomal amidase activity was highest in rat lung and lowest in the liver (per mg protein). Glutathione S-aryl transferase activity was highest in the liver. The in vitro metabolism of carbaryl, phosphamidon, and chlorotoluron by the various centrifugal fractions revealed many differences. Carbaryl metabolism was greater in the liver microsomal fractions than in any other preparation. 1-Naphthol was the major metabolite in all tissue fractions. Although very little metabolism of phosphamidon occurred in the rat, metabolism in the rat liver postmitochondrial fraction was slightly higher with respect to the production of metabolites than in the supernatant and microsomes combined. Chlorotoluron was not metabolized by any of the tissue fractions of the rat. At least a low level of activity toward some compounds was observed in all tissues, but this study confirmed that the liver was the most active metabolizing tissue as well as having the highest levels of enzymatic activity usually associated with pesticide metabolism.     

DDT effects on certain ATP related systems in the peripheral nervous system of the lobster Homarus americanus

Effects of DDT (1,1,1-trichloro-2,2-bis-(p-chlorophenyl) ethane) on various ATP utilizing enzymes in the lobster peripheral nerve were studied. On the basis of inhibition by ouabain and DDT, four classes of ATPase enzymes were recognized. They are: (1) ATPase activity that is sensitive to both ouabain and DDT inhibition, or Type A, (2) ATPase activity that is sensitive to DDT inhibition only, or Type B, (3) ATPase activity that is sensitive to ouabain only, and (4) ATPase activity that is not sensitive to either ouabain or DDT. The Type A ATPase is considered to be a part of the total (Na⁺K⁺) ATPase enzyme associated with the electrogenic pump. The Type B ATPase consisted of an uncharacterized Na⁺, K⁺, and Mg²⁺ stimulated ATPase and includes also a small portion of Mg²⁺ stimulated ATPase. Ca²⁺ stimulated ATPase activity was also detected but was not significantly affected by DDT. Proteins with actomyosin-like properties were also recognized to be present, though this superprecipitation process was only slightly affected by DDT.Other systems studied include the transfer of (γ-³²P) ATP to endogenous proteins and added histone in the presence and absence of c-AMP. DDT generally stimulated the process of ³²P incorporation, while it inhibited a portion of the specific c-AMP dependent protein kinase activity.It was concluded from these studies that DDT has a potential to inhibit or otherwise interfere with a variety of enzymatic reactions that utilize ATP as a substrate. Of these systems, the Type B ATPase bore overall resemblance to the possible target for DDT.     

Isolation of insect microsomal oxidases by rapid centrifugation

Only about 60% of the total relative gravitational force conventionally used to sediment microsomes is needed to prepare highly active microsomes from the midgut tissues of an insect larva. A rapid preliminary centrifugation for 2 min at 39,000g_max effectively removed contaminating microorganisms, tissue debris, nuclei, and mitochondria. The supernatant was recentrifuged for 20 min to 210,000g to sediment the microsomes. There were no losses of microsomal oxidase activities or degradation of cytochrome P-450 to the inactive form (P-420) resulting from the application of the higher gravitational force. Incorporation of 1 mM EDTA in the buffer and washing the microsomes resulted in an improved yield of the cytochrome compared to that in microsomes prepared in sucrose. Yields of microsomal protein, cytochrome P-450, and NADPH-cytochrome c reductase in the rapidly isolated microsomes were as good as those in conventionally prepared microsomes. The apparent kinetic characteristics of several microsomal oxidation activities and optical difference spectra of Types 1 and 2 ligands were identical in the rapidly and conventionally prepared microsomes. The morphological appearance of the microsomes was examined by electron microscopy. Microsomal pellets prepared by either method were indistinguishable. The rapid procedure saves significant time in microsome preparation and yields microsomal oxidase activities as good or slightly better than those prepared by usual centrifuged procedures.     

Selective inhibition of separate esterases in rat and mouse liver microsomes hydrolyzing malathion,trans-permethrin,and cis-permethrin

Separate esterase activities of rat and mouse liver microsomes hydrolyzing malathion, trans-permethrin, and cis-permethrin were differentiated on the basis of their sensitivities to inhibition by paraoxon and α-naphthyl N-propylcarbamate (NPC). In rat liver microsomes, the malathionhydrolyzing activity was more sensitive to both inhibitors and showed a different time course of NPC inhibition than the activities hydrolyzing the permethrin isomers. Paraoxon completely inhibited trans-permethrin hydrolysis, but only partially inhibited that of cis-permethrin. The paraoxonsensitive trans- and cis-permethrin-hydrolyzing activities were not differentially inhibited, but separate inhibition curves were obtained for the inhibition of trans- and cis-permethrin hydrolysis by NPC. The mouse liver esterase activity hydrolyzing trans-permethrin showed a similar paraoxon sensitivity to that of rat liver, but that the paraoxon-sensitive portion of the cis-permethrinhydrolyzing activity was 5.5-fold less sensitive to paraoxon than the corresponding rat liver activity and was clearly differentiated from the mouse liver trans-permethrin-hydrolyzing activity. The mouse liver malathion-hydrolyzing activity was 100-fold less sensitive to paraoxon and 14-fold less sensitive to NPC than the corresponding rat liver activity. Rat and mouse liver esterase activities hydrolyzed trans- and cis-permethrin at similar rates under standard assay conditions, but mouse liver esterases were 10-fold less active in hydrolyzing malathion. The higher specific activity of rat liver malathion-hydrolyzing esterases resulted from the greater apparent affinity and maximum velocity for malathion hydrolysis. These results demonstrate that the hydrolysis of malathion, trans-permethrin, and cis-permethrin by rat and mouse liver microsomal preparations involves several esterases with differing substrate specificities and inhibitor sensitivities.     

The role of tyrosinase in the inactivation of house fly microsomal mixed-function oxidases

The low mixed-function oxidase activity of house fly microsomes has been associated with low cytochrome P-450 content and NADPH-cytochrome c reductase activity. The microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity could be decreased by the addition of catechol and increased by the addition of cyanide to the homogenates. Similar results were obtained with rat liver microsomes treated with tyrosinase and catechol. During the inactivation of rat liver microsomal enzymes by tyrosinase and catechol, crosslinking of microsomal proteins occurred. These results suggest that the instability of house fly microsomal mixed-function oxidase may be due in part to the action of contaminating tyrosinase on endogenous substrates.     

Effect of insecticides on the short-circuit current and resistance of isolated frog skin

The effects of aldrin, carbaryl, α- and γ-chlordane, dichlorodiphenyldichloroethane (DDD), dichlorodiphenyltrichloroethane (DDT), dieldrin, endrin, heptachlor, lindane, methoxychlor, and nonachlor on the short-circuit current and resistance of the isolated intact frog skin were studied. The short-circuit current is approximately equivalent to the rate of active transport of sodium, while the resistance indicates the summed ionic permeability of the skin. At a concentration of 2 × 10^?4M, only carbaryl, DDD, dieldrin, and lindane produced significant (P<0.05, paired t test) changes in the short-circuit current. Nonachlor produced a decrease (P=0.12) in the short-circuit current and also increased the resistance (P=0.07). DDD, dieldrin, and carbaryl caused significant increases in short-circuit current while at the same time the resistance was significantly decreased. Lindane increased both the short-circuit current and the resistance. It was concluded that the frog skin probably contains effective permeability barriers that prevent externally applied insecticides from reaching the site of active sodium transport. It appeared likely that most of the insecticides produced the observed effects on the frog skin by altering the sodium permeability of the outer barrier.     

DDT and sodium transport in the eel electroplaque

DDT inhibits the ATPase activity of the intact eel electroplaque. At a concentration of 10^?5M, DDT inhibited 46% of the total ATPase activity, and 10^?4M DDT inhibited 62% of the total ATPase activity and 62% of the ouabain-sensitive ATPase activity. The latter concentration of DDT reduced the rate of Na efflux from intact electroplaques and slowed the rate of recovery of the membrane potential following a large depolarization produced by carbamylcholine application. Repetitive direct stimulation of the innervated membrane at 10 Hz during the application of 10^?4M DDT produced a significant irreversible depolarization. Ouabain, 10^?4M, produced similar effects. The possible role of the inhibition of active NaK transport in producing the symptoms of DDT poisoning is discussed.