Dichlorodiphenylacetic Acid Research Articles

Two-dimensional chromatography for enantiomeric analysis of mitotane and its metabolite o,p′-DDA in patients with adrenocortical carcinoma indicates enantioselective metabolism

Mitotane is a chiral drug used to treat adrenocortical carcinoma, being metabolized to the o,p’-dichlorodiphenyl acetic acid (o,p’-DDA), also a chiral compound. Despite of its therapeutic significance, the overall ratios and enantiomers have not been known. In this study, we analyzed the enantiomers of mitotane and o,p’-DDA in the plasma of patients by a newly developed chiral-phase method employed in two-dimensional chromatography. Important differences were observed in the ratio of (S)/(R)-mitotane, which varied substantially from 1:1.2 to 1:10 whereas the (S)/(R)-o,p’-DDA ratio was relatively conserved, at approximately 2:1. These findings provide evidence for the enantioselective metabolism and provide a method for further analyses of mitotane and metabolites, which can explain the variation in the therapeutic response.

Formation and Fate of Point-Source Nonextractable DDT-Related Compounds on Their Environmental Aquatic-Terrestrial Pathway.

Nonextractable residues (NER) are pollutants incorporated into the matrix of natural solid matter via different binding mechanisms. They can become bioavailable or remobilize during physical-chemical changes of the surrounding conditions and should thus not be neglected in environmental risk assessment. Sediments, soils, and groundwater sludge contaminated with DDXs (DDT, dichlorodiphenyltrichloroethane; and its metabolites) were treated with solvent extraction, sequential chemical degradation, and thermochemolysis to study the fate of NER-DDX along different environmental aquatic-terrestrial pathways. The results showed that DDT and its first degradation products, DDD (dichlorodiphenyldichloroethane) and DDE (dichlorodiphenyldichloroethylene), were dominant in the free extractable fraction, whereas DDM (dichlorodiphenylmethane), DBP (dichlorobenzophenone), and DDA (dichlorodiphenylacetic acid) were observed primarily after chemical degradation. The detection of DDA, DDMUBr (bis( p-chlorophenyl)-bromoethylene), DDPU (bis( p-chlorophenyl)-propene) and DDPS (bis( p-chlorophenyl)-propane) after chemical treatments evidenced the covalent bindings between these DDXs and the organic matrix. The identified NER-DDXs were categorized into three groups according to the three-step degradation process of DDT. Their distribution along the different pathways demonstrated significant specificity. Based on the obtained results, a conceptual model of the fate of NER-DDXs on their different environmental aquatic-terrestrial pathways is proposed. This model provides basic knowledge for risk assessment and remediation of both extractable and nonextractable DDT-related contaminations.

Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism.

Three point mutations R335S, L336V and V476L, distinguish the sequence of a cytochrome P450 CYP6A2 variant assumed to be responsible for 1,1,1-trichloro-2,2-bis-(4'-chlorophenyl)ethane (DDT) resistance in the RDDT(R) strain of Drosophila melanogaster. To determine the impact of each mutation on the function of CYP6A2, the wild-type enzyme (CYP6A2wt) of Cyp6a2 was expressed in Escherichia coli as well as three variants carrying a single mutation, the double mutant CYP6A2vSV and the triple mutant CYP6A2vSVL. All CYP6A2 variants were less stable than the CYP6A2wt protein. Two activities enhanced in the RDDT(R) strain were measured with all recombinant proteins, namely testosterone hydroxylation and DDT metabolism. Testosterone was hydroxylated at the 2beta position with little quantitative variation among the variants. In contrast, metabolism of DDT was strongly affected by the mutations. The CYP6A2vSVL enzyme had an enhanced metabolism of DDT, producing dicofol, dichlorodiphenyldichloroethane and dichlorodiphenyl acetic acid. The apparent affinity of the enzymes CYP6A2wt and CYP6A2vSVL for DDT and testosterone was not significantly different as revealed by the type I difference spectra. Sequence alignments with CYP102A1 provided clues to the positions of the amino acids mutated in CYP6A2. These mutations were found spatially clustered in the vicinity of the distal end of helix I relative to the substrate recognition valley. Thus this area, including helix J, is important for the structure and activity of CYP6A2. Furthermore, we show here that point mutations in a cytochrome P450 can have a prominent role in insecticide resistance.

Gas chromatographic-electron capture determination of 2,4′-dichlorodiphenylacetic acid from in-vitro adrenal transformations of mitotane and its analogs

Gas chromatographic-electron capture determination of 2,4′-dichlorodiphenylacetic acid from in-vitro adrenal transformations of mitotane and its analogs

Metabolic Activation and Binding of Mitotane in Adrenal Cortex Homogenates

Mitotane [1‐(2‐chlorophenyl)‐1‐(4‐chlorophenyl)‐2,2‐dichloroethane,o,p′‐DDD] is an adrenocorticolytic agent of value in the treatment of adrenocortical carcinoma and Cushing's syndrome. In support of a program to develop agents superior to mitotane, it is the purpose of this study to explore the relationship of the metabolism of mitotane to its binding to adrenal cortex tissue from several sources. The objective was to detect the mitotane moiety responsible for its covalent binding in various test systems. Studies were conducted with an125I‐Iabeled analog of mitotane, 1‐(2‐chlorophenyl)‐1‐(4‐iodophenyl)‐2,2‐dichloroethane, prior to a comparison to results with lower specific activity [14C]mitotane. With dog adrenal cortical whole homogenates, the majority of covalent binding was to proteins with an additional one‐sixth of the total bound radioactivity associated with a phospholipid fraction. No radioactivity was associated with DNA. The rank order of species in regard to metabolism and protein binding was bovine > dog > rat adrenal homogenates > human normal adrenal or tumor homogenates. The percentage of radioactivity recovered from the hydrolysates of those fractions was uniformly high. In addition, the only metabolite present in the hydrolysates corresponded to 1‐(2‐chlorophenyl)‐1‐(4‐iodophenyl)acetic acid from the iodo analog ofo,p′‐DDD and the correspondingo,p′‐dichlorodiphenylacetic acid (o,p′‐DDA) fromo,p′‐DDD. Our results are consistent with an acyl chloride being the reactive intermediate formed from the dichloromethyl moiety of mitotane, which leads to both DDA metabolite formation and binding to adrenal cortical bionucleophiles.

Identification of o,p'-Dichlorodiphenyl Acetic Acid as a Urinary Metabolite of 1-(o-Chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane

Identification of o,p'-Dichlorodiphenyl Acetic Acid as a Urinary Metabolite of 1-(o-Chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane

Urinary excretion of insecticide metabolites. Excretion of para-nitrophenol and DDA as indicators of exposure to parathion.

The urinary excretion of p-nitrophenol and of dichlorodiphenylacetic acid (DDA) was measured in spraymen applying fo·rmulations containing both parathion and DDT In each instance, excretory level of metabolite correlated well with exposure to the pesticide. Para-nitrophenol excretion levels showed changes of greater magnitude in response to a specific exposure than did DDA excretion. However, in general the excretory patterns for p-nitrophenol and DCA were similar. Both metabolites reached peak levels at about the same time interval (p-nitrophenol, 8m7 hours; DCA, 10m1 hours) after exposure beganm Excretion of both p-nitrophenol and DDA tended to increase with an increase in ambient temperature. Excretion curves for both metabolites showed diurnal variation during the period after exposure had ended. On the average for a specific exposure, more parathion-derived material than DDTderived material was recovered in the urine.

Biodegradation of Dichlorodiphenyltrichloroethane: Intermediates in Dichlorodiphenylacetic Acid Metabolism by Aerobacter aerogenes

Biodegradation of Dichlorodiphenyltrichloroethane: Intermediates in Dichlorodiphenylacetic Acid Metabolism by <i>Aerobacter aerogenes</i>

Use of a sensitive new method for determination of the urinary excretion of dichlorodiphenylacetic acid (DDA) by human subjects

A simple procedure for the isolation and determination of p-dichlorodiphenylacetic acid (DDA) in urine and other biological fluids has been presented. The method, utilizing inexpensive laboratory apparatus, commercially available Florisil, and prepared thin-layer chromatography sheets, permits the identification and estimation of DDA in the order of 0.005 ppm. The average recovery of from 5 to 50 μg DDA added to DDA-free urine was 68.4%. Urinary DDA excretion of 13 human subjects picked at random from the general population was found to average 46 μg/day, ranging from 13 to 117 μg/day.

Gytotoxicity of Dichlorodiphenylacetic Acid (DDA) Upon Cultured KB and HeLa Cells, and its Reversal by Mevalonic Acid

SummaryDichlorodiphenylacetic acid (DDA) (50 μg/ml) completely disrupted the growth of cultured KB and HeLa cells. Mevalonic acid (25 μg/ml) provided essentially complete protection against the cytox-icity of 50 μg/ml of DDA. Neither acetate nor cholesterol afforded protection against the DDA effect.

Biodegradation of Dichlorodiphenyltrichloroethane: Intermediates in Dichlorodiphenylacetic Acid Metabolism by Aerobacter aerogenes

Biodegradation of Dichlorodiphenyltrichloroethane: Intermediates in Dichlorodiphenylacetic Acid Metabolism by Aerobacter aerogenes

Studies of choline acetylase inhibition by DDA (dichlorodiphenylacetic acid) and its possible relationship to DDT toxicity

The effect of DDA, a metabolite of DDT, on rabbit intestinal motility in vitro, on the patellar reflex, and on a choline acetylase system was studied. Na-DDA was found to inhibit the pendular activity of rabbit small intestine at a minimal bath concentration of 10 −4 M. Exposure to 10 −3 M DDA completely blocked rhythmic activity and depressed intestinal tone. The activity could be restored by washing or by exposure to 10 −8 acetylcholine, 5 × 10 −8 physostigmine, or 2 × 10 −8 TEPP. The induction of peristaltic activity by physostigmine or TEPP was partially inhibited or completely blocked by prior treatment with DDA. A method for the injection of drugs directly to the lumbar spinal cord via a lumbar artery is described. DDA injected by this route in a coconut oil emulsion in doses between 0.4 and 1.8 mg facilitated the patellar reflex response. Doses larger than 2 mg depressed the patellar reflex. When the patellar reflex was depressed, spontaneous activity could be momentarily restored by acetylcholine or acetylcholine + TEPP similarly injected. Pantothenyl alcohol by this route gave a weak restoration of the reflex response. Diphenylylethylacetic acid and diphenylylamylacetic acid, known choline acetylation inhibitors, produced effects similar to those of DDA. Acetylcholine synthesis by an in vitro system utilizing acetone-dried brain powder as a source of choline acetylase was depressed by Na-DDA. When Ac-CoA was used as a substitute for ATP + CoA in this system, the level of acetylcholine synthesis was diminished but there was little inhibition by Na-DDA. The results indicate that the acetate activating step is the principal site of the block. The effects of DDA on intestinal motility and the patellar reflex are interpreted on the basis of inhibition of the acetate-activating step. Other alternative possible hypotheses are discussed. The possible relationship of these findings to the problem of DDT intoxication is discussed.