Decreased inhibitory activity of salt wash preparations toward aminoacyl-tRNA binding to liver ribosomes in vitro after administration of DDT

Ribosomes were prepared from the microsome fraction of a liver homogenate of control and DDT-treated adult male rats using Triton X-100, as well as by several other methods. In every case, a 0.5 M salt wash fraction of the ribosomes from control animals contained an inhibitor of peptide synthesis which exerted its action on the ribosomes by reducing the elongation factor 1-stimulated step measured in vitro, i.e., aminoacyl-tRNA binding. The inhibitory activity was removed by heat treatment and trypsin digestion and by Pronase digestion. The inhibitor was different from the thiol and GTP-sensitive inhibitor of liver microsomal membranes and from the hemin-reversible inhibitor of reticulocyte lysates, but it was similar to the ribosomal inhibitor described for certain nonmammalian cells. In contrast to the situation during liver regeneration and during the response to aminonucleoside nephrosis, no inhibitory activity could be detected in the salt wash fraction after the administration of DDT. These results emphasize the necessity for a salt-wash step in the preparation of ribosomes for measurement of their activity after the induction of microsomal enzymes.     

Residues of DDT in cottonseed after spraying with DDT and torbidan

DDT at 1.12 and 2.24 kg/ha a.i. and Torbidan at 5 and 10 litre formulation/ha (1 and 2 kg DDT/ha) were sprayed five times on cotton over a period of 15 weeks. Seeds from the first pick of the crop were found to contain as residues pp′-DDT and pp′-DDE [1,1-dichloro-2,2-di- (4-chlorophenyl)ethylene]. The highest residue level (0.783 parts/10⁶) was found in seeds from Torbidan 10 litre/ha treatment.     

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.     

Metabolism of four resmethrin isomers by liver microsomes

Microsomal esterases of mouse and rat liver readily cleave the trans- but not the cis-isomers of resmethrin (5-benzyl-3-furylmethyl chrysanthemate). The ester linkage also appears to undergo oxidative cleavage when esterase attack is minimal, i.e., with (+)-cis- and particularly (?)-cis-resmethrin in microsome-NADPH systems and with any of the isomers when NADPH is added to microsomes pretreated with TEPP. Metabolites retaining the ester linkage are detected in significant amounts only with (+)-cis-resmethrin in which case they are formed by oxidation at either the trans(E)- or cis(Z)-methyl group of the isobutenyl moiety with or without oxidation of the benzylfurylmethyl group. Metabolites of each acid moiety include chrysanthemic acid and up to six derivatives of this acid formed by oxidation at the trans(E)- or cis(Z)-methyl group yielding the corresponding alcohol, aldehyde, or acid, with chrysanthemate isomer and enzyme source variations in the preferred site of oxidation. The major identified metabolite of the alcohol moiety is either benzylfurylmethanol or the corresponding carboxylic acid depending on the enzyme system used. In the course of microsomal oxidation, a fragment from the alcohol but not the acid moiety of (+)-trans- and (+)-cis-resmethrin is strongly bound to microsomal components. These findings confirm in vivo studies on the isomeric variations in metabolism of the resmethrin components.     

Species and sex differences in the reductive dechlorination of DDT by supplemented liver preparations

p,p′-DDT was converted to DDD, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane, by unheated 12,000g supernate supplemented with NADPH, made from liver of rat, mouse, hamster, quail, chicken and pigeon. The additional presence of exogenous riboflavin did not augment reduction. If, however, riboflavin was added to unheated microsomes supplemented with NADPH or NADH the rate of reduction was more than doubled.Heated 12,000g supernate, unsupplemented or containing only exogenous riboflavin, did not reduce DDT. When NAD(P)H was present, big species differences in activity occurred, the pigeon and cockerel scarcely reducing any DDT in 2 hr, the Wistar rat supernate being most active. Addition to heated 12,000g supernate of 30 μg riboflavin as well as NAD(P)H resulted in increased reductive activity for all six species. The species difference in effect of exogenous riboflavin between the pigeon and rat was also observed using four DDT analogs as substrates. A sex-related difference in activity of preheated, supplemented 12,000g supernate from livers of the chicken and Norwegian hooded rat was not found when unheated preparations were used. In contrast to the activity possessed by preheated 12,000g supernate of rat liver supplemented with NAD(P)H, similarly supplemented preheated microsomes did not reduce DDT. On adding riboflavin as well as NAD(P)H, however, preheated hepatic microsomes of both pigeon and rat produced DDD, and this activity was further increased by addition of unheated microsomal (105,000g) supernate. The biocatalytic system functioning in preheated preparations appears to need at least one component of heated microsomes, NAD(P)H and one or more water-soluble components.     

Substrate-specificity and toxicological significance of pyrethroid-hydrolyzing esterases of mouse liver microsomes

An esterase or esterases in acetone powder preparations of mouse liver microsomes hydrolyze the cyclopropanecarboxylate ester linkage of pyrethroid insecticide chemicals derived from primary alcohols. The rate of cleavage of (+)-trans-chrysanthemates with various alcohol moieties decreases in the following order: 5-propargyl-2-furylmethyl; 5-benzyl-3-furylmethyl (bioresmethrin); 3-phenoxybenzyl; tetrahydrophthalimidomethyl esters. The hydrolysis rate of benzylfurylmethyl esters with various acid moieties decreases in the order: (+)- or (?)-trans-chrysanthemate; (+)-trans-ethanochrysanthemate; tetramethylcyclopropanecarboxylate; (+)- or (?)-cis-chrysanthemate or (+)-cis-ethanochrysanthemate. The trans-isomers of chrysanthemates and ethanochrysanthemates are hydrolyzed from 2.6- to more than 50-fold more rapidly than the corresponding cis-isomers. This enzyme system does not hydrolyze secondary alcohol esters, i.e., allethronyl (+)-trans- and (+)-cis-chrysanthemates.On intraperitoneal administration to mice, the (+)-trans-chrysanthemate and -ethanochrysanthemate of benzylfurylmethanol are of very low toxicity relative to the corresponding (+)-cis-isomers and the tetramethylcyclopropanecarboxylate. S,S,S-tributyl phosphorotrithioate (DEF) pretreatment increases the toxicity of these five compounds by 2.6- to more than 188-fold, with the exception of bioresmethrin whose toxicity is not altered. When the toxicity is increased, it is probably the result of esterase inhibition since DEF strongly inhibits the esterase activity of fresh liver microsomes while the mixed-function oxidase system remains active. The oxidase system metabolizes the chrysanthemates more rapidly than the ethanochrysanthemates of benzylfuryl-methanol. Depending upon the pyrethroid involved, the esterase or the mixed-function oxidase system, or both may be responsible for limiting the toxicity of these pyrethroids to mice.     

Effect of s-triazine and phenoxyalkanoic acid herbicides on UDP-glucuronosyltransferase in rat liver microsomes

Twelve herbicides, representatives of two chemical groups, substituted phenoxyalkanoic acids and s-triazines, were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol (4-NP), phenolphthalein (PPh) and 4-methylumbelliferone (4-MU) by rat liver microsomes. One millimole MCPA, ametryn and cyanazine significantly decreased PPh UDP-glucuronosyltransferase (UGT) activity, while propazine was found to be a most potent inhibitor of 4-NP glucuronidation. Concentrations of 0.1 mM dichlorprop and cyanazine were still inhibitory against PPh-UGT. The inhibition of 4-MU glucuronidation by the herbicides was low and not specific. As a whole, s-triazine derivatives were more inhibitory than the substituted phenoxyalkanoic acids. Kinetic studies with propazine revealed a non-competitive type of inhibition towards the acceptor substrate 4-NP, with an apparent K_i value of 0.540 mM . With ametryn, an uncompetitive type of inhibition against PPh and a mixed type of inhibition towards UDPGA were found, with apparent K_i values of 0.330 mM and 0.380 mM , respectively. © 1999 Society of Chemical Industry     

Effects of added protein (bovine serum albumin) on the rate and enantiotopic selectivity of oxidative O-demethylation of methoxychlor in rat liver microsomes

The addition of bovine serum albumin (BSA) to rat liver microsomal suspensions during the O-demethylation of methoxychlor (1,1-bis(4-methoxyphenyl)-2,2,2-trichloroethane) produced a good substrate concentration-reaction rate relationship, and allowed us to determine the rate parameters for this reaction. The apparent K_m became larger when the albumin content was increased, whereas V_max was not changed. The enantiotopic selectivity of the reaction dramatically changed when the substrate concentration was increased in the absence of BSA in phenobarbital-treated rat liver microsomes.     

Effects of reducing DDT usage on total DDT storage in humans

Agricultural uses of the insecticide DDT were cancelled by the U.S. Environmental Protection Agency December 31, 1972. However, the domestic use of DDT had begun to decline before this action. Beginning July 1969, residues of DDT and its metabolites were measured in human adipose tissue collected through an annual national survey. Levels of total DDT equivalent residues in human adipose have decreased slightly, but the frequencies of finding DDT or its metabolites have remained high. The most marked decline in residue concentration has been found in the youngest age group (0-14 years). Approximately 80 percent of the total DDT equivalent found in this survey was DDE. These data show that the reduction of the agricultural uses of DDT has decreased human exposure to and storage of this chemical.     

The inhibition of HEOM epoxide hydrase in mammalian liver microsomes and insect pupal homogenates

A range of compounds were tested as inhibitors of the enzyme epoxide hydrase, using a cyclodiene epoxide (HEOM) as substrate. Rat and rabbit liver microsomes and pupal homogenates of the blowfly (Calliphora erythrocephala) and the yellow mealworm (Tenebrio molitor) were compared as sources of the enzyme. Only minor differences were found between the four enzyme preparations, when considering I₅₀ values and percentage inhibition at standard concentration. The simple epoxide 1,1,1-trichloropropane-2,3-epoxide and two glycidyl ethers p-nitrophenyl glycidyl ether and p-ethylphenyl glycidyl ether tended to have lower I₅₀ values (1.8×10^?6 to 8.0×10^?5M) than triphenyl phosphate and SKF 525A (4.5×10^?5 to 1.4×10^?4M). Triphenyl phosphate and SKF 525A were competitive inhibitors for both the rat and Tenebrio enzymes. The only clear difference found between these two epoxide hydrase preparations was with respect to their inhibition by 1,1,1-trichloropropane-2,3-epoxide, which was an uncompetitive inhibitor with the rat enzyme, but showed kinetics of mixed inhibition with the insect preparation.     

Metabolism of trifluralin,profluralin, and fluchloralin by rat liver microsomes

Three structurally related [¹⁴C]dinitroaniline herbicides, trifluralin, profluralin, and fluchloralin, were extensively metabolized in vitro by both normal and phenobarbital-induced rat liver microsomes. Identification of the metabolites in the ethyl acetate extracts indicated that aliphatic hydroxylation, N-dealkylation, reduction of a nitro group, and cyclization were the predominant metabolic routes for these herbicides in vitro. Of particular interest was the formation of a benzimidazole metabolite.     

Selective alterations of membrane properties of cultured human liver cells caused by the insecticide DDT

A variety of membrane-specific parameters was examined in both intact cells and isolated plasma membranes following exposure of cultured human liver cells to the insecticide 1,1-(2,2,2-trichloroethylidene)bis(4-chloro)benzene (DDT). Uptake of DDT was at equilibrium within 6 hr. In contrast, a decrease in the number of β-adrenergic hormone receptors first became significant after 48 hr of cell exposure. Whereas the uptake was largely reversible, the loss in the number of β receptors did not recover after DDT-exposed cells were cultured in fresh medium lacking the insecticide. Experiments in vitro substantiated the time lag of the biological effect. The decrease in receptor proteins was persistent in membranes with increased phospholipid unsaturation. Temperature-activity profiles (“Arrhenius plots”) of $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}\text{-ATPase}$ and 5′-nucleotidase were unchanged. Endogenous tryptophan fluorescence of membrane proteins was lower in membranes from DDT-exposed cells. These selective alterations in membrane parameters suggest a specific interaction of DDT with membrane proteins; interference with cellular protein synthesis is possible. The results indicate that membrane lipid “fluidization” does not play a physiologically important role in the mechanism of DDT action in biomembranes.     

Comparative excretion of DDT analogues into milk of indian buffalo,Bubalus bubalis L. following oral administration

Four groups of Indian buffaloes were fed daily with 25 mg of p,p′-DDT p,p′-TDE p,p′-DDE or o,p′-DDT for 100 days. Milk was analysed for organochlorine residues during this period and also for 100 days after pesticide administration had been discontinued. For the period showing ‘plateau level’ residues, 17.2% of p,p′-DDE, 17% of p,p′-TDE, 14% of p,p′-DDT as p,p′-DDT (3.5%); p,p′-TDE (10.5%); 3.2% of p,p′-DDT as o,p′-DDT (1.3%) and o,p'-TDE (1.9%) of their administered amounts were excreted in the milk. Since these compounds were excreted at different rates, the residue levels in the milk expected from a given feed would depend on their concentration and proportional distribution in the feed. The maximum tolerable content of DDT analogues in feed was derived to be 0.1 mg kg^?1 (dry weight basis) by using the maximum accumulation coefficient and incorporation of the necessary safety margin. It is concluded that Indian buffaloes fed with rations contaminated with a total of DDT analogues below this limit will yield milk of acceptable quality. Following the termination of feeding with contaminated rations, the elimination of p,p′-DDE in the milk took the longest time and that of o,p′-DDT the shortest. These results suggest that the time required for the initial high residue concentration to decline to less than the legal limit would be determined by the relative amounts of DDE, TDE and DDT in the milk, after elimination of the potent source of contamination.     

Metabolism of cisanilide (Cis-2,5-dimethyl-1-pyrrolidinecarboxanilide) by rat liver microsomes

Metabolism of [phenyl-¹⁴C] and [(2,5) pyrrolidine-¹⁴C] cisanilide was investigated in vitro with microsomal preparations from rat liver. Microsomal activity was associated with a mixed-function oxidase system that required O₂ and NADPH and was inhibited by CO. Two major ether-soluble metabolites were isolated. They were identified as primary oxidation products: 2-hydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide (A) and 4′-hydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide (B). Minor ether-soluble metabolites were also isolated. Precursor product studies and qualitative thin layer chromatography analysis of [pyrrolidine-¹⁴C] and methylated [phenyl-¹⁴C] hydrolysis products suggested that these metabolites were secondary oxidation products formed from metabolites A or B. One of these metabolites appeared to be the dihydroxy product 2,4′-dihydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide. Crude microsomal preparations (postmitochondrial supernatant fractions) also formed small quantities (<10%) of polar metabolites. Enzyme hydrolysis with β-glucuronidase (Escherichia coli) indicated that approximately 50% of these metabolites were glucuronides. Similarities and differences in cisanilide oxidation in vivo in plants and in vitro with rat liver microsomal preparations were discussed.     

糠菌唑在大鼠、小鼠、兔、狗和人肝微粒体中的立体选择性代谢

采用高效液相色谱-串联质谱 (HPLC-MS/MS) 手性色谱柱法定量分析肝微粒体中的糠菌唑,通过密度泛函理论计算光谱与振动圆二色光谱 (VCD) 和红外光谱 (IR) 比对,确定了糠菌唑4种对映体的绝对构型;以大鼠、小鼠、兔、狗和人肝微粒体为模型,研究了糠菌唑的立体选择性降解行为。结果表明:在供试肝微粒体中,4种异构体的降解均遵循一级反应动力学方程,且在人和小鼠肝微粒体中的代谢速率相对较慢。(2S, 4R)-和 (2R, 4S)-糠菌唑在5种供试肝微粒体中的立体选择性趋势一致,而 (2R, 4R)- 和 (2S, 4S)-糠菌唑只在兔肝微粒体中的降解有显著的立体选择性差异,在小鼠肝微粒体中几乎没有立体选择性。酶促反应动力学结果也证实了糠菌唑代谢的立体选择性,并显示 (2R, 4R)- 和 (2S, 4S)-糠菌唑在供试肝微粒体中的酶促反应趋势存在种属差异。     

FAC1基因对禾谷镰刀菌的菌丝膨大处细胞核分裂的影响

 与大多数丝状真菌一样,禾谷镰刀菌中也存在依赖cAMP信号调节的蛋白激酶A(protein kinase A,PKA)信号通路-cAMP-PKA信号通路。前期研究发现,该信号通路在脱氧雪腐镰刀菌烯醇(Deoxynivalenol, DON)的合成中扮演着重要角色。FAC1作为cAMP-PKA信号通路上游基因,它的缺失可导致DON无法合成。本研究发现,FAC1的缺失可严重影响DON合成相关基因TRI1在转录水平和翻译水平上的表达。显微观察发现,Tri1定位在细胞核周围,并且与野生型菌株PH-1相比,fac1突变体在产毒培养基内菌丝膨大结构的形成增多。此外,与未膨大菌丝相比,野生型菌株PH-1菌丝膨大结构处细胞核分裂明显增多,该位点多于4个细胞核的比例达到65.4%,而fac1突变体中菌丝膨大结构处核分裂数仅为0~3个,其中0~2个的占90.7%。研究结果表明,在产毒培养基中特有形成的菌丝膨大结构与DON合成之间并没有必然联系, DON毒素的合成应与菌丝膨大结构处细胞核分裂密切相关。