Carbohydrate metabolism dynamic in chlorpropham- and 1,4-dimethylnaphthalene-treated potatoes and its effect on the browning of French fries.

The use of a sprout suppressor is crucial for the use of potatoes beyond their natural dormancy period. The main sprout inhibitor used on a commercial scale, chlorpropham (CIPC), is becoming increasingly limited owing to its toxicity. Therefore, we evaluated the effectiveness of 1,4-dimethylnaphthalene (1,4-DMN) compared to CIPC in controlling sprouting and maintaining the quality of potato, Solanum tuberosum 'Asterix', during cold storage. Treatment with 1,4-DMN reduced fresh weight loss and controlled the number and length of sprouts comparable to CIPC. Compared to the control, both sprouting inhibitors led to higher starch and lower reducing sugar contents, and the tubers retained the recommended quality for industrial processing. After frying, less browning was observed in French fries obtained from 1,4-DMN- or CIPC-treated tubers. We ascertain that 1,4-DMN besides being an efficient sprouting inhibitor and alternative to CIPC, it contributes to maintaining the quality of French fries after cold storage.

Chlorpropham, a carbamate ester herbicide, has an endocrine-disrupting potential by inhibiting the homodimerization of human androgen receptor.

This study was carried out to provide the evidence with respect to the adverse potential of chlorpropham, a representative carbamate ester herbicide product, on the endocrine system by using in vitro testing methods in accordance with the Organization for Economic Cooperation and Development Test Guideline No. 458 (22Rv1/MMTV_GR-KO human androgen receptor [AR] transcriptional activation assay) and a bioluminescence resonance energy transfer-based AR homodimerization assay. Results revealed that chlorpropham had no AR agonistic effects, but it was determined to be a true AR antagonist without intrinsic toxicity against the applied cell lines. In the mechanism of chlorpropham-induced AR-mediated adverse effects, chlorpropham suppressed cytoplasmic AR translocation to the nucleus by inhibiting the homodimerization of the activated ARs. This suggests that chlorpropham exposure caused endocrine-disrupting effects through its interactions with human AR. Additionally, this study might help identify the genomic pathway of the AR-mediated endocrine-disrupting potential of N-phenyl carbamate herbicides.

The essential cysteines in the CIPC motif of the thioredoxin-like Trypanosoma brucei MICOS subunit TbMic20 do not form an intramolecular disulfide bridge in vivo.

The mitochondrial protein import machinery of trypanosomatids is highly divergent from that of the well-studied models such as baker's yeast. A notable example is that the central catalyst of the mitochondrial intermembrane space import and assembly pathway (MIA), named Mia40, is missing in trypanosomatids. Mia40 works in a two-step process. First it recognizes by direct binding reduced MIA substrate proteins and then catalyzes their oxidative folding to produce intramolecular disulfide bridges. It was recently proposed that a thioredoxin-like subunit of the trypanosomal mitochondrial contact site and cristae organizing system (MICOS) called TbMic20 may be the Mia40 replacement. Our study performed on procyclic stage of the parasite revealed that each of the two cysteines in TbMic20's active site is essential for the stability of MIA substrate proteins although they do not form a disulfide bridge in vivo. The two cysteines of Mia40's active site form an intramolecular disulfide bridge at steady state, which is a prerequisite for its oxidative folding of MIA substrates. Thus, we conclude that TbMic20 is unlikely to represent a bona fide Mia40 replacement and plays a still unresolved role in the stability and/or import of MIA substrates in trypanosomatids. Despite this, the effect of TbMic20 depletion and mutation indicates that the trypanosomal MICOS complex still plays a vital role in the maturation and/or stability of proteins imported by the MIA pathway.

CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the putative Drosophila ortholog of CLOCK INTERACTING PROTEIN CIRCADIAN.

The Drosophila circadian clock is driven by a transcriptional feedback loop in which CLOCK-CYCLE (CLK-CYC) binds E-boxes to transcribe genes encoding the PERIOD-TIMELESS (PER-TIM) repressor, which releases CLK-CYC from E-boxes to inhibit transcription. CLOCKWORK ORANGE (CWO) reinforces PER-TIM repression by binding E-boxes to maintain PER-TIM bound CLK-CYC off DNA, but also promotes CLK-CYC transcription through an unknown mechanism. To determine how CWO activates CLK-CYC transcription, we identified CWO target genes that are upregulated in the absence of CWO repression, conserved in mammals, and preferentially expressed in brain pacemaker neurons. Among the genes identified was a putative ortholog of mouse Clock Interacting Protein Circadian (Cipc), which represses CLOCK-BMAL1 transcription. Reducing or eliminating Drosophila Cipc expression shortens period, while overexpressing Cipc lengthens period, which is consistent with previous work showing that Drosophila Cipc represses CLK-CYC transcription in S2 cells. Cipc represses CLK-CYC transcription in vivo, but not uniformly, as per is strongly repressed, tim less so, and vri hardly at all. Long period rhythms in cwo mutant flies are largely rescued when Cipc expression is reduced or eliminated, indicating that increased Cipc expression mediates the period lengthening of cwo mutants. Consistent with this behavioral rescue, eliminating Cipc rescues the decreased CLK-CYC transcription in cwo mutant flies, where per is strongly rescued, tim is moderately rescued, and vri shows little rescue. These results suggest a mechanism for CWO-dependent CLK-CYC activation: CWO inhibition of CIPC repression promotes CLK-CYC transcription. This mechanism may be conserved since cwo and Cipc perform analogous roles in the mammalian circadian clock.

Mineralization of the herbicide swep by a two-strain consortium and characterization of a new amidase for hydrolyzing swep.

BACKGROUND: Swep is an excellent carbamate herbicide that kills weeds by interfering with metabolic processes and inhibiting cell division at the growth point. Due to the large amount of use, swep residues in soil and water not only cause environmental pollution but also accumulate through the food chain, ultimately pose a threat to human health. This herbicide is degraded in soil mainly by microbial activity, but no studies on the biotransformation of swep have been reported. RESULTS: In this study, a consortium consisting of two bacterial strains, Comamonas sp. SWP-3 and Alicycliphilus sp. PH-34, was enriched from a contaminated soil sample and shown to be capable of mineralizing swep. Swep was first transformed by Comamonas sp. SWP-3 to the intermediate 3,4-dichloroaniline (3,4-DCA), after which 3,4-DCA was mineralized by Alicycliphilus sp. PH-34. An amidase gene, designated as ppa, responsible for the transformation of swep into 3,4-DCA was cloned from strain SWP-3. The expressed Ppa protein efficiently hydrolyzed swep and a number of other structural analogues, such as propanil, chlorpropham and propham. Ppa shared less than 50% identity with previously reported arylamidases and displayed maximal activity at 30 °C and pH 8.6. Gly449 and Val266 were confirmed by sequential error prone PCR to be the key catalytic sites for Ppa in the conversion of swep. CONCLUSIONS: These results provide additional microbial resources for the potential remediation of swep-contaminated sites and add new insights into the catalytic mechanism of amidase in the hydrolysis of swep.

Biodegradation of chlorpropham and its major products by Bacillus licheniformis NKC-1.

Chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] (CIPC), an important phenyl carbamate herbicide, has been used as a plant growth regulator and potato sprout suppressant (Solanum tuberosum L) during long-term storage. A bacterium capable of utilizing the residual herbicide CIPC as a sole source of carbon and energy was isolated from herbicide-contaminated soil samples employing selective enrichment method. The isolated bacterial strain was identified as Bacillus licheniformis NKC-1 on the basis of its morphological, cultural, biochemical characteristics and also by phylogenetic analysis based on 16S rRNA gene sequences. The organism degraded CIPC through its initial hydrolysis by CIPC hydrolase enzyme to yield 3-chloroaniline (3-CA) as a major metabolic product. An inducible 3-CA dioxygenase not only catalyzes the incorporation of molecular oxygen but also removes the amino group by the deamination yielding a monochlorinated catechol. Further, degradation of 4-chlorocatechol proceeded via ortho- ring cleavage through the maleylacetate process. 3-Chloroaniline and 4-chlorocatechol are the intermediates in the CIPC degradation which suggested that dechlorination had occurred after the aromatic ring cleavage. The presence of these metabolites has been confirmed by using ultra-violet (UV), high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), Fourier transmission-infrared (FT-IR), proton nuclear magnetic resonance (1H NMR) and gas chromatography-mass (GC-MS) spectral analysis. Enzyme activities of CIPC hydrolase, 3-CA dioxygenase and chlorocatechol 1, 2-dioxygenase were detected in the cell-free-extract of the CIPC culture and are induced by cells of NKC-1 strain. These results demonstrate the biodegradation pathways of herbicide CIPC and promote the potential use of NKC-1 strain to bioremediate CIPC-contaminated environment with subsequent release of ammonia, chloride ions and carbon dioxide.

Spatial heterogeneity in degradation characteristics and microbial community composition of pesticide biopurification systems.

AIMS: To investigate spatial and temporal differences in degradation characteristics and microbial community composition of pesticide biopurification systems. METHODS AND RESULTS: Pilot-scale biofilters were supplemented with the potato-sprouting suppressant chloropropham. Two biofilters were inoculated with a chloropropham-degrading mixed culture, while the other two were not inoculated. Biodegradation rate, size and composition of the microbial community were monitored during 72 days at different biofilter depths. First of all, results showed that inoculation was not necessary to obtain efficient degradation although it shortens the biofilter's start-up period. Secondly, a higher biodegradation rate and chloropropham- and 3-chloroaniline-degrading microbial community size could be seen in the top part of the inoculated as well as the noninoculated biofilters. Finally, analysis of the microbial community composition shows that no clear spatial stratification of the microbial community could be found in any biofilter. However, the microbial diversity increases over time in all biofilters and on all biofilter depths, suggesting that during the time of the experiment, the biofilters develop a broad carrying capacity in which a genetically very diverse range of chloropropham- and 3-chloroaniline-degrading species can thrive. CONCLUSIONS: In this study, a vertical gradient of the chloropropham- and 3-chloroaniline-degrading community composition, in terms of density and temporal and spatial diversity, was clearly established and was directly connected to a vertical gradient of chloropropham biodegradation activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The major part of degradation activity takes place in the top part of the biofilter, suggesting that it could be possible to use shorter biofilter reactors or higher loading rates to treat chloropropham waste streams, making this type of bioremediation technique economically more feasible.

Inclusion complexation of chloropropham with ß-cyclodextrin: preparation, characterization and molecular modeling.

An inclusion complex between the agrochemical chloropropham (CIPC) and ß-cyclodextrin (ß-CD) was prepared. A 2:1 host-guest stoichiometry was conformed by elemental analysis. From the phase solubility studies, the calculated stepwise stability constants were K(1)=224.6L/mol and K(2)=939.2L/mol, respectively. FT-IR, thermoanalysis and (1)H NMR spectra were applied to characterize the complex. It was speculated that the inclusion mode was two ß-CD cavities included the chlorophenyl and the isopropyl moiety of one CIPC molecule, which was in agreement with the most predominant configuration optimized by molecular modeling. By complexation with ß-CD, the water solubility and the thermal stability of CIPC were prominently improved.

Planktonic versus biofilm catabolic communities: importance of the biofilm for species selection and pesticide degradation.

Chloropropham-degrading cultures were obtained from sludge and soil samples by using two different enrichment techniques: (i) planktonic enrichments in shaken liquid medium and (ii) biofilm enrichments on two types of solid matrixes (plastic chips and gravel). Denaturing gradient gel electrophoresis fingerprinting showed that planktonic and biofilm cultures had a different community composition depending on the presence and type of added solid matrix during enrichment. This was reflected in the unique chloropropham-degrading species that could be isolated from the different cultures. Planktonic and biofilm cultures also differed in chloropropham-degrading activity. With biofilm cultures, slower chloropropham removal was observed, but with less build-up of the toxic intermediate 3-chloroaniline. Disruption of the biofilm architecture resulted in degradation characteristics shifting toward those of the free suspensions, indicating the importance of a well-established biofilm structure for good performance. These results show that biofilm-mediated enrichment techniques can be used to select for pollutant-degrading microorganisms that like to proliferate in a biofilm and that cannot be isolated using conventional shaken-liquid procedures. Furthermore, the influence of the biofilm architecture on the pesticide degradation characteristics suggests that for bioaugmentation the use of biofilm catabolic communities might be a proficient alternative to using planktonic freely suspended cultures.

Level and fate of chlorpropham in potatoes during storage and processing.

Chlorpropham (isopropyl 3-chlorocarbanilate) is a pesticide used to control sprouting of potatoes during long-term storage. The objective of the present study was to establish the total chlorpropham residue balance (residues in unwashed and washed whole tubers, peeled tubers, peels, boiled and pureed tubers, and washing and cooking waters) for two potato varieties after uniform application as a function of storage time under different typical storage conditions (in a basement, storehouse, and refrigerator) in Lithuania. Chlorpropham concentration on washed and unwashed tubers decreased from approximately 15 mg kg(- 1) after storage for 28 d to approximately 9 mg kg(- 1) after storage for 85 d. Peel concentrations decreased from approximately 50 mg kg(- 1) at 5 d after treatment to approximately 20 mg kg(- 1) at 85 d after treatment. The average concentration in the two varieties of peeled tubers in the three storage facilities was 1 mg kg(- 1). Chlorpropham concentrations in the wash water decreased from 3.5 to 1.0 mg L(- 1) when the storage time increased from 28 to 85 d. The cooking water concentration similarly decreased, from > 0.2 mg L(- 1) at 28 d after treatment to > 0.1 mg L(- 1) at 85 d after treatment. The results of this study show that chlorpropham concentrations in tubers under these typical conditions were below the recently revised levels that are acceptable for residues in whole potatoes (30 mg kg(- 1)) and peels (40 mg kg(- 1)). Also, despite fluctuating conditions during storage, chlorpropham treated tubers did not sprout, as compared to untreated tubers, which sprouted.

Biotransformation of chlorpropham (CIPC) in isolated rat hepatocytes and xenoestrogenic activity of CIPC and its metabolites by in vitro assays.

1: The metabolism and action of chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC, a post-harvest agent) were studied in freshly isolated rat hepatocytes, and the oestrogen-like activity of CIPC and its metabolites was assessed by in vitro assays. The exposure of hepatocyte suspensions to CIPC caused concentration- (0.25-1.0 mM) and time- (0-3 h) dependent cell death, which was assessed by Trypan blue exclusion, accompanied by losses of cellular adenosine triphosphate and adenine nucleotide pools, and formation of cell bleb. 2: CIPC at a weakly toxic level (0.25 or 0.5 mM) was metabolized to isopropyl N-(3-chloro-4-hydroxyphenyl)carbamate (4OH-CIPC) and subsequently to its glucuronide and sulfate conjugates (major metabolites) or alternatively to the minor metabolites 3-chloroaniline (3CA) and 3-chloroacetanilide. CIPC (0.25 mM) added to hepatocyte suspensions was distributed equally between hepatocytes and the extracellular medium during the incubation. The glucuronide rather than the sulfate conjugate of 4OH-CIPC predominantly increased in the medium with time, while the amount of unconjugated free 4OH-CIPC in the extracellular medium increased by approximately threefold compared with the amount in the cell fraction after 0.5 h and then decreased rapidly accompanied by increases in the conjugates. This indicates that unconjugated free 4OH-CIPC produced in hepatocytes was temporarily excreted in the extracellular medium and subsequently converted to the conjugates via re-influx into hepatocytes. 3: Diethylstilbestrol (DES), bisphenol A (BPA) and 4-hydroxybenzoic acid butyl ester (butylparaben), which are known xenoestrogenic compounds, competitively displaced 17beta-oestradiol bound to the oestrogen receptor-alpha (ERalpha) in a concentration-dependent manner; IC50 values of DES, BPA, butylparaben and its derivative 3-chloro-4-hydroxybenzoic acid butyl ester (3-chloro-butylparaben) were approximately 10(-8), 10(-5), 5 x 10(-5) and 5 x 10(-4) M, respectively. In contrast, neither CIPC nor 4OH-CIPC impaired the binding of 17beta-oestradiol to ERalpha at concentrations ranging from 10(-9) to 10(-4) M, whereas at concentrations of >5 x 10(-4) M, the binding affinity of 4OH-CIPC was greater than that of CIPC. In a proliferation assay of MCF-7 cells, CIPC, 4OH-CIPC and 3CA did not increase cell numbers at concentrations ranging from 10(-9) to 10(-5) M, but these compounds at a concentration of 10(-4) M induced a considerable decrease in cell numbers relative to the control. The results suggest that even if CIPC is metabolized to 4OH-CIPC by hepatocytes, the chlorine adjacent to the 4-hydroxy group added to the intermediate as well as 3-chloro-butylparaben obstructs the appearance of oestrogen-like effects via an interaction between the intermediate and the ER.

Chlorpropham induces mitochondrial dysfunction in rat hepatocytes.

The metabolism and action of chlorpropham (isopropyl N-(3-chlorophenyl)carbamate; CIPC, a post-harvest agent) and its metabolites were studied in freshly isolated rat hepatocytes and isolated rat hepatic mitochondria, respectively. The exposure of hepatocytes to CIPC caused a concentration (0.25-1.0 mM)- and time (0-3h)-dependent cell death accompanied by loss of cellular ATP and adenine nucleotides. CIPC at a weakly toxic level (0.5 mM) was metabolized to isopropyl N-(3-chloro-4-hydroxyphenyl)carbamate (4OH-CIPC) and subsequently to its glucuronide and sulfate conjugates (major metabolites) or alternatively to a minor metabolite 3-chloroaniline (3CA). The addition of SKF-525A (50 microM), an inhibitor of microsomal monooxygenase, enhanced the CIPC (0.5 mM)-induced cytotoxicity accompanied by loss of ATP and 4OH-CIPC and inhibited the decrease in the concentration of the parent compound. CIPC led to a strong decrease in cellular ATP content compared to its metabolites, 4OH-CIPC and 3CA. On the other hand, the exposure of isolated hepatic mitochondria to CIPC reduced State 3 respiration with a FAD-linked substrate (succinate plus rotenone) and/or with a NAD+ -linked substrate (pyruvate plus malate), whereas State 3 respiration with ascorbate plus tetramethyl-p-phenylendiamine (cytochrome oxidase-linked respiration) was not affected markedly by CIPC. Further, the addition of CIPC caused an increase in the rate of State 4 oxygen consumption, indicating an uncoupling effect, and a decrease in the rate of State 3 oxygen consumption in a concentration-dependent manner, respectively. In contrast, the addition of neither 4OH-CIPC nor 3CA markedly affected the rate of states 3 and/or 4 oxygen consumption. These results indicate that CIPC-induced cytotoxicity is mediated by the parent compound rather than by its metabolites such as 4OH-CIPC and 3CA, and that the toxicity is associated with a rapid depletion of ATP via impairment of mitochondrial function related to oxidative phosphorylation.

Study of potatoes' sprout inhibitor treatments with chlorprophame.

Studies carried out in 1999 by the University of Ghent showed that 36% of potatoes' samples contained Chlorprophame (CIPC) residues and that 7.9% of them exceeded the maximal limit of residues (RML), fixed at 5 ppm. The heterogeneity of sprout inhibitor application would be one of the causes of over-dosage. However, this heterogeneity would also cause under-dosages leading to problems when controlling the sprouting in potatoes stored over 6 degrees C. This study aims at determining some technical causes of the heterogeneity of CIPC sprout inhibitor treatments when storing potatoes. The study concerns two treatment techniques: dusting and spraying. To draw up an inventory of mechanical treatments in Belgium, a survey has been conducted among 28 farmers throughout Belgium. 35 samples have been taken at random in the different storage rooms to analyse the content of CIPC residue. In order to do so, a method of analysis: the gas chromatography in capillary phase with detection by mass spectrophotometry, has been developed. Tests have been carried out by changing several parameters such as the material, the product or the place in the storage line, in order to assess the CIPC application techniques. The survey made it possible to analyse qualitatively, from the declarations of farmers, the causes of heterogeneity linked to treatment techniques. An almost systematically over-dosage of the CIPC quantity has been noticed. However, out of the 35 samples analysed, only 2 had residue contents higher than the RML. The comparative analysis of the quantities applied and the residues contained in the samples made it possible to quantify the heterogeneity of the applications depending on the techniques. The tests carried out show in a general way that mechanical dusting, even though having a less constant flowrate than sprayers, leads to less important variation of the residue between samples. In testing conditions, the heterogeneity of the antigerminative treatment decreases when applied by means of a mechanical duster. In practise, these results are distorted by topical applications of CIPC. The combination of this practise with a too high heterogeneity of the treatment are to be avoided in order to have a good preservation and meet the residues standards.

Metabolism and cytotoxicity of chlorpropham (CIPC) and its essential metabolites in isolated rat hepatocytes during a partial inhibition of sulphation and glucuronidation reactions: a comparative study.

The changes in metabolism and cytotoxicity of chlorpropham (CIPC) and its major metabolites, 4-hydroxychlorpropham (4-OH CIPC), 3-chloroaniline, and 3-chloroacetanilide were investigated in isolated rat hepatocyte suspensions after a partial inhibition of sulphation and glucuronidation and the two reactions combined in an attempt to assess the part of each of them in the enhanced CIPC toxicity observed in vivo after D-galactosamine treatment. With sulphation and glucuronidation effective, CIPC has a cytolytic effect and reduces intracellular ATP and K+ level while 4-OH CIPC has a weak cytolytic effect but modifies ATP and K+ level in a greater extent than CIPC. Inhibition of sulphation does not affect the cytotoxicity of CIPC or 4-OH CIPC because there is a compensatory increase in the amount of 4-OH CIPC glucuronide formed and the level of free 4-OH CIPC always remain low. In contrast, when incubations are carried out with either CIPC or 4-OH CIPC, the presence of D-galactosamine leads to a decrease of glucuronide and sulphate conjugates accompanied, respectively, by a 3.6-fold and 6. 9-fold increase of the free 4-OH CIPC level in the culture medium. This alteration of the metabolism is followed by a marked reduction of ATP synthesis with a concomitant modification of cell permeability. The cytolytic effect is due to CIPC itself, whereas the effect on energy supply was attributed to free 4-OH CIPC. The results demonstrate a combined effect of free 4-OH CIPC and D-galactosamine on intracellular ATP level that could account for the partial inhibition of sulphation. This change in the CIPC metabolism could explain the increased CIPC toxicity observed in vivo after D-galactosamine pretreatment.

The carry-through of residues of thiabendazole, tecnazene and chlorpropham from potatoes following manufacture into potato crisps and jacket potato crisps.

Potatoes, commercially treated with thiabendazole, tecnazene and chlorpropham, were processed into potato crisps and jacket potato crisps at a crisp factory using standard manufacturing conditions. A multi-residue method based on gas chromatography with mass spectrometric detection was developed and used to determine pesticide residue levels in the potatoes and potato crisps. Results showed that the residues of all three pesticides were significantly reduced to less than 2% and less than 10% of the maximum theoretical residue carry-through level for potato crisps and jacket potato crisps respectively.

Effects of modulation of sulphation and glucuronidation on chlorpropham metabolism and cytotoxicity in isolated rat hepatocytes.

After modulation of sulphation and glucuronidation, the relationship between the changes in metabolism and cytotoxicity of chloropropham (CIPC), a widely used herbicide, was investigated in isolated rat hepatocyte suspensions. Under physiological conditions, CIPC had a cytolytic effect, modified membrane permeability and reduced intracellular ATP level. CIPC was metabolized by hepatocytes mainly into 4-OH chlorpropham sulphate (37%) and glucuronide conjugates (18%). Inhibition of sulphation, by omitting sulphate from the isolation and incubation media, did not affect the cytotoxicity of CIPC, since there was a 2.5-fold compensatory increase in 4-OH CIPC glucuronide. Inhibition of glucuronidation by adding 4 mM D-galactosamine in the incubation medium led to a 66% decrease of glucuronide conjugate and simultaneously to a 32% decrease of sulphate conjugate. In that case, concentrations of free 4-OH CIPC in both hepatocytes and incubation medium were markedly increased, while those of 3-chloroaniline and 3-chloroacetanilide were slightly modified and remained low. This alteration of metabolism was accompanied by modification of cell permeability and reduction in ATP synthesis. The cytolytic effect was due to CIPC itself, whereas the effect on energetic metabolism was attributed to a metabolite. Results demonstrated for the first time a partial inhibition of sulphation by D-galactosamine (4 mM), probably due to the effect of D-galactosamine on intracellular ATP levels.

Mechanism of cadmium-decreased glucuronidation in the rat.

In isolated rat hepatocytes, cadmium (0-200 microM) decreased the overall glucuronidation of both isopropyl N-(3-chloro-4 hydroxyphenyl)carbamate (4-hydroxychlorpropham, 4-OHCIPC) and 4-nitrophenol in a concentration-dependent manner. In contrast, in native rat liver microsomes, glucuronidation of 4-OHCIPC was increased by cadmium through activation of microsomal 4-OHCIPC glucuronosyl transferase. In addition, in rat microsome incubations, the net amount of 4-OHCIPC glucuronide was also indirectly increased by cadmium through a reduction in the activity of beta-glucuronidase. As the effect of cadmium on the activity of 4-OHCIPC glucuronosyl transferase could not account for the decrease in glucuronide formation in intact hepatocytes, the influence of cadmium on the availability of UDP-glucuronic acid (UDPGA) was investigated further. In isolated rat hepatocytes, cadmium depleted the UDPGA content in a dose-dependent manner without a change in the UDP glucose (UDPG) content. Cadmium did not increase the breakdown of UDPGA by microsomal UDPGA pyrophosphatase but strongly decreased (30-66%) the synthesis of the cofactor in the cytosol by inhibiting UDP-glucose dehydrogenase (UDPGDH). Cadmium (10-50 microM) was found to inhibit the purified enzyme from bovine liver (EC 1.1.1.22) non-competitively. In vivo in the absence of a substrate undergoing glucuronidation, cadmium administration, 1.5 and 2.5 mg Cd/kg i.v., to normally fed rats resulted in a 15 and 30% decrease of hepatic UDPGA, respectively. However, in the liver, neither the NAD+/NADH ratio nor the UDPG content was significantly changed following cadmium treatment. Both in vitro and in vivo results support the conclusion that in intact cells the reduction in overall 4-OHCIPC glucuronidation caused by cadmium was due to a decrease in UDPGA availability which results from the inhibiting effect of cadmium on UDPGDH.

Cadmium-induced alterations of chlorpropham metabolism in isolated rat hepatocytes.

In order to investigate the various steps of chlorpropham (CIPC) metabolism which could be influenced by cadmium, isolated rat hepatocytes were incubated in the presence of CIPC (0.1 mM) and of increasing Cd concentrations (0-180 microM). The results showed that Cd accumulation in hepatocytes was in good correlation to its concentration in the incubation medium. At 90 microM Cd, hydroxylation of CIPC was only slightly decreased by 30%, while CIPC hydrolysis into 3-chloraniline was unaffected by the presence of Cd. Accordingly, unchanged CIPC increased in hepatocytes. At 27 microM Cd, free 4-hydroxychlorpropham (4-OHCIPC) increased in the intracellular medium as a consequence of a strong suppression of both sulfation and glucuronidation which was related to the strong depletion of the intracellular ATP level under the combined influences of both cadmium and free 4-OHCIPC. Acetylation of 3-chloroaniline, which represents a minor pathway of CIPC metabolism, was already markedly suppressed (43%) with the lowest Cd concentration (27 microM). These in vitro results suggest that Phase II reactions are more sensitive to Cd than Phase I processes and that Cd enhanced the CIPC cytotoxicity as shown by alterations of the membrane integrity.