Rapid partial degradation of DDT by a cytochrome P‐450 model system

Abstract

Heme compounds, in combination with a reducing agent and oxygen, can express various activities of cytochrome P-450 enzymes. In the present study it was found that a mixture of hemin and excess cysteine was able to degrade the insecticide DDT partially. The major products were three water-soluble, non-toxic conjugates of DDT metabolites with cysteine which had lost two or three of the five chlorine atoms of DDT per molecule and whose structures were elucidated by gas chromatography/mass spectrometry. In 0.05 M NH4HC03, pH 7.7/ ethanol ( 5 : 6 , by vol.), the degradation reaction catalyzed by the hemin-cysteine model system was at least 8 x lo4 times Faster than the uncatalyzed reaction. In the presence of a designed 24-residues polypeptide or p-casein, two DDT-binding proteins, an additional fourfold increase in the rate of DDT degradation was observed. Although the concentrations of DDT and cysteine occurring in an organism would be expected to be lower than those in the experiments described, the formation of water-soluble conjugates of DDT metabolites with cysteine (and other amino acids) could also play a role in metabolism and excretion of DDT in vivo.The objective of the present investigation was to develop a model system for DDT degradation and to study the influence of DDT-binding proteins, in particular a designed 24-residue polypeptide [l - [4]. Aerobic reactions catalyzed by (inducible) cytochrome P-450 enzymes yield mainly DDA [his@-chloropheny1)acetic acid, Fig. I], the principal urinary excretion product of DDT in mammals, and DCBP (p,p'-dichlorobenzophenone, Fig. 1) [5, 61. Conjugates of DDA were also isolated. One such product was found to be a covalent complex of DDA, aspartic acid, and serine obtained after administration of DDT or DDA in rats [7].The prosthetic group of cytochrome P-450 enzymes is heme whose ligation with a thiolate seems to be essential for activity [S, 91. It is known that the biological activities of these enzymes can be mimicked by model systems. A hemin-cysteine complex, for example, was shown to catalyze the hydroxylation of aniline, p-toluidine and other substrates in the presence of excess cysteine [lo -121; hematin, myoglobin, and hemoglobin were able to reduce alkyl halides to the corresponding alkanes in the presence of sodium dithionite [13,14].Correspondence to B. Gutte, Biochemisches Institut der Universitat Zurich, Winterthurerstrasse 190, CH-8057 Zurich, SwitzerlandAbbreviations. DDT, t,1,1 -trichloro-2,2-bis(p-chlorophenyl)-ethane; DDE, 1,1-dichloro-2,2-bis(pp-chlorophenyl)ethylene; DDD, 1 ,I -dichloro-2,2-bis(p-chlorophenyl)ethane; DDA, bis(p-chloropheny1)acetic acid; DCBP, p,p'-dichlorobenzophenone; Fmoc, fluorenylmethyloxycarbonyl; GC-MS, gas chromatography/mass spectrometry.Enzymes. Cytochrome P-450 enzymes (EC 1.14.14.1); /I-Dgalactosidase (EC 3.2.1.23).We found that the mixture of hemin and excess cysteine also mediated the degradation of DDT and that the rate of this reaction was increased by addition of a designed 24-residue DDT-binding polypeptide [l...

Serum Folate and DDT Isomers and Metabolites Are Inversely Associated in Chinese Women: A Cross-Sectional Analysis

“…Strong inverse associations were observed between cysteine and pp′DDT, pp′DDE, pp′DDD, and op′DDT. Consistent with the present findings, Langen et al [16] showed in vitro that a mixture of hemin and excess cysteine was able to partially degrade DDT. Although the concentration of cysteine in tissues is much lower than the concentration used in this in vitro system, this finding suggests that cysteine, along with other amino acids, could influence the metabolism and excretion of DDT in vivo .…”

“…In addition, indole-3-carbinol, a component of cruciferous vegetables, alters DDT metabolism and reduces its estrogenic effects by favoring metabolism through the 2-hydroxyoestone pathway, a more benign pathway with regard to breast cancer risk compared to the 16-alpha-hydroxyoestrone pathway [15]. Langen et al [16] showed that a mixture of hemin and excess cysteine was able to degrade DDT partially, where the degradation reaction catalyzed by the hemin-cysteine model was 8 × 10 4 times faster than the uncatalyzed reaction and produced 3 water-soluble, nontoxic, cysteine-containing conjugates. Other animal studies suggest that nutrient levels may be influenced by xenobiotics.…”

A P450 gene associated with robust resistance to DDT in ciliated protozoan, Tetrahymena thermophila by efficient degradation

“…In other organisms cytochrome P450 enzymes anchored on the endoplasmic reticulum participate in the detoxification of xenobiotics, drug, and pesticide metabolism (Lah et al, 2011;Langen et al, 1989;Werck-Reichhart and Feyereisen, 2000). Results of computational analyses combined with subcellular localization of CYP5013C2-GFP suggested that CYP5013C2 was a typical endoplasmic reticulum-anchored protein like CYP3A4 in Saccharomyces cerevisiae (Liao et al, 2006) .…”

Fusion Proteins from Artificial and Natural Structural Modules

“…Interestingly, of all the mixtures of natural proteins with hemin and cysteine tested for their ability to degrade DDT only a mixture containing ß-casein had an activity comparable to that of DBP, hemin and cysteine [23] the main reason for this probably being the relatively strong binding of both DDT and hemin by ß-casein (K D , 5.5x10 -5 M and 5x10 -5 M, respectively). Because of its broad spectrum of biological functions (nutrient, casomorphin activity [29], DDT binding etc.)…”

“…4a) whose affinity for the insecticide (K D , 9x10 -7 M in buffer/ethanol, 1:1) was >1000 times higher than that of serum albumin, a well-known carrier of apolar metabolic intermediates and xenobiotics in the circulation. Together with hemin and excess cysteine, this peptide accelerated DDT degradation reactions by a factor of approximately 3.5x10 5 as compared to the rate of uncatalyzed degradation [23]. In the mixture of DDT with DBP, hemin and cysteine, DBP improved the solubility of DDT for the degradation reactions catalyzed by the hemincysteine cytochrome P-450 model system [24].…”