Identification of genes showing altered DNA methylation and gene expression in the renal proximal tubular cells of rats treated with ochratoxin A for 13 weeks.

Ochratoxin A (OTA) is a mycotoxin that causes renal carcinogenicity following the induction of karyomegaly in proximal tubular cells after repeated administration to rats. Here, we performed gene profiling regarding altered DNA methylation and gene expression in the renal tubules focusing on the mechanism of OTA-induced carcinogenesis. For this purpose, OTA or 3-chloro-1,2-propanediol (3-MCPD), a renal carcinogen not inducing karyomegaly, was administered to rats for 13 weeks, and DNA methylation array and RNA sequencing analyses were performed on proximal tubular cells. Genes for which OTA altered the methylation status and gene expression level, after excluding genes showing similar expression changes by 3-MCPD, were subjected to confirmation analysis of the transcript level by real-time reverse-transcription PCR. Gene Ontology (GO)-based functional annotation analysis of validated genes revealed a cluster of hypermethylated and downregulated genes enriched under the GO term "mitochondrion," such as those associated with metabolic reprogramming in carcinogenic process (Clpx, Mrpl54, Mrps34, and Slc25a23). GO terms enriched for hypomethylated and upregulated genes included "response to arsenic-containing substance," represented by Cdkn1a involved in cell cycle arrest, and "positive regulation of IL-17 production," represented by Osm potentiating cell proliferation promotion. Other genes that did not cluster under any GO term included Lrrc14 involved in NF-κB-mediated inflammation, Gen1 linked to DNA repair, Has1 related to chromosomal aberration, and Anxa3 involved in tumor development and progression. In conclusion, a variety of genes engaged in carcinogenic processes were obtained by epigenetic gene profiling in rat renal tubular cells specific to OTA treatment for 13 weeks.

Exploring the thermostable properties of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by a combinatorial directed evolution strategy.

As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained. Those mutation sites were subsequently combined by adopting several rounds of iterative saturation mutagenesis (ISM) approach. After four rounds of saturation mutagenesis, one best mutant ISM-4 with a 3400-fold improvement in half-life (t 1/2) inactivation at 65 °C, 18 °C increase in apparent T m value, and 20 °C increase in optimum temperature was obtained, compared to wild-type HheC. To the best of our knowledge, the mutant represents the most thermostable HheC variant reported up to now. Moreover, the mutant was as active as wild-type enzyme for the substrate 1,3-dichloro-2-propanol, and they remained most enantioselectivity of wild-type enzyme in the kinetic resolution of rac-2-chloro-1-phenolethanol, exhibiting a great potential for industrial applications. Our structural investigation highlights that surface loop regions are hot spots for modulating the thermostability of HheC, the residues located at these regions contribute to the thermostability of HheC in a cooperative way, and protein rigidity and oligomeric interface connections contribute to the thermostability of HheC. All of these essential factors could be used for further design of an even more thermostable HheC, which, in turn, could greatly facilitate the application of the enzyme as a biocatalyst.

3-Chloro-1,2-propanediol biodegradation by Ca-alginate immobilized Pseudomonas putida DSM 437 cells applying different processes: mass transfer effects.

3-Chloro-1,2-propanediol (3-CPD) biodegradation by Ca-alginate immobilized Pseudomonas putida cells was performed in batch system, continuous stirred tank reactor (CSTR), and packed-bed reactor (PBR). Batch system exhibited higher biodegradation rates and 3-CPD uptakes compared to CSTR and PBR. The two continuous systems (CSTR and PBR) when compared at 200 mg/L 3-CPD in the inlet exhibited the same removal of 3-CPD at steady state. External mass-transfer limitations are found negligible at all systems examined, since the observable modulus for external mass transfer Ω â‰ª 1 and the Biot number Bi > 1. Intra-particle diffusion resistance had a significant effect on 3-CPD biodegradation in all systems studied, but to a different extent. Thiele modulus was in the range of 2.5 in batch system, but it was increased at 11 when increasing cell loading in the beads, thus lowering significantly the respective effectiveness factor. Comparing the systems at the same cell loading in the beads PBR was less affected by internal diffusional limitations compared to CSTR and batch system, and, as a result, exhibited the highest overall effectiveness factor.

Orally administered glycidol and its fatty acid esters as well as 3-MCPD fatty acid esters are metabolized to 3-MCPD in the F344 rat.

IARC has classified glycidol and 3-monochloropropane-1,2-diol (3-MCPD) as group 2A and 2B, respectively. Their esters are generated in foodstuffs during processing and there are concerns that they may be hydrolyzed to the carcinogenic forms in vivo. Thus, we conducted two studies. In the first, we administered glycidol and 3-MCPD and associated esters (glycidol oleate: GO, glycidol linoleate: GL, 3-MCPD dipalmitate: CDP, 3-MCPD monopalmitate: CMP, 3-MCPD dioleate: CDO) to male F344 rats by single oral gavage. After 30 min, 3-MCPD was detected in serum from all groups. Glycidol was detected in serum from the rats given glycidol or GL and CDP and CDO in serum from rats given these compounds. In the second, we examined if metabolism occurs on simple reaction with rat intestinal contents (gastric, duodenal and cecal contents) from male F344 gpt delta rats. Newly produced 3-MCPD was detected in all gut contents incubated with the three 3-MCPD fatty acid esters and in gastric and duodenal contents incubated with glycidol and in duodenal and cecal contents incubated with GO. Although our observation was performed at 1 time point, the results showed that not only 3-MCPD esters but also glycidol and glycidol esters are metabolized into 3-MCPD in the rat.

Formation of epichlorohydrin, a known rodent carcinogen, following oral administration of 1,3-dichloro-2-propanol in rats.

The observed toxicity and carcinogenicity of 1,3-dichloro-2-propanol (DCP) in rodents is thought to be due to the formation of reactive metabolites, epichlorohydrin (ECH) and dichloroacetone (DCA). However, there is no direct evidence for the formation of these metabolites from exposure to DCP in rodents due to the challenges of measuring these reactive intermediates directly in vivo. The objective of this work was to investigate the metabolism of DCP to ECH and DCA in vivo by first developing a sensitive analytical method in a suitable biological matrix and analyzing samples from rats administered DCP. DCA reacted rapidly in vitro in rat blood, plasma, and liver homogenate, precluding its detection. Because ECH rapidly disappeared in liver homogenate, but was relatively long-lived in plasma and blood in vitro, blood was selected for analysis of this metabolite. Following a single oral dose of 50 mg/kg DCP in male or female Harlan Sprague-Dawley rats, ECH was detected in blood with a maximum concentration reached at ≤13.7 min. ECH was cleared rapidly with a half-life of ca. 33 and 48 min in males and females, respectively. Following a single oral dose of 25 mg/kg ECH in male and female rats, the elimination half-life of ECH was ca. 34 and 20 min, respectively; the oral bioavailability of ECH was low (males, 5.2%; females, 2.1%), suggesting extensive first pass metabolism of ECH following oral administration. The area under the concentration vs time curve for ECH following oral administration of DCP and intravenous administration of ECH was used to estimate the percent of the DCP dose converted to ECH in rats. On the basis of this analysis, we concluded that in male and female rats following oral administration of 50 mg/kg DCP, ≥1.26% or ≥1.78% of the administered dose was metabolized to ECH, respectively.

Expression, characterization, and improvement of a newly cloned halohydrin dehalogenase from Agrobacterium tumefaciens and its application in production of epichlorohydrin.

A gene encoding halohydrin dehalogenase (HHDH) from Agrobacterium tumefaciens CCTCC M 87071 was cloned and expressed in Escherichia coli. To increase activity and stability of HHDH, 14 amino acid residues around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected as targets for site-directed mutagenesis. The studies showed that the mutant HHDH (Mut-HHDH) enzyme had a more accessible substrate-binding pocket than the wild-type HHDH (Wt-HHDH). Molecular docking revealed that the distance between the substrate and active site was closer in mutant which improved the catalytic activity. The expressed Wt-HHDH and Mut-HHDH were purified and characterized using 1,3-dichloro-2-propanol (1,3-DCP) as substrates. The specific activity of the mutant was enhanced 26-fold and the value of k cat was 18.4-fold as compared to the Wt-HHDH, respectively. The Mut-HHDH showed threefold extension of half-life at 45 °C than that of Wt-HHDH. Therefore it is possible to add 1,3-DCP concentration up to 100 mM and epichlorohydrin (ECH) was produced at a relatively high conversion and yield (59.6 %) using Mut-HHDH as catalyst. This Mut-HHDH could be a potential candidate for the upscale production of ECH.

Enhanced biotransformation of 1,3-dichloro-2-propanol to epichlorohydrin via resin-based in situ product removal process.

Biotransformation of 1,3-dichloro-2-propanol (DCP) to epichlorohydrin (ECH) by the whole cells of recombinant Escherichia coli expressing halohydrin dehalogenase was limited by product inhibition. To solve this problem and improve the ECH yield, a biotransformation strategy using resin-based in situ product removal (ISPR) was investigated. Seven macroporous resins were examined to adsorb ECH: resin HZD-9 was the best. When 10 % (w/v) HZD-9 was added to batch biotransformation, 53.3 mM ECH was obtained with a molar yield of 88.3 %. The supplement of the HZD-9 increased the ECH volumetric productivity from 0.5 to 2.8 mmol/l min compared to without addition of resin. In fed-batch biotransformation, this approach increased ECH from 31 to 87 mM. These results provide a promising basis for the biosynthesis of ECH.

3-MCPD Induced Mitochondrial Damage of Renal Cells Via the Rhythmic Protein BMAL1 Targeting SIRT3/SOD2.

Biorhythm regulates a variety of physiological functions and enables organisms to adapt to changing environments. 3-Monochloro-1,2-propanediol (3-MCPD) is a common food thermal processing contaminant, and the kidney is its toxic target organ. However, the nephrotoxicity mechanism of 3-MCPD has not been fully elucidated. In the study, we found that 3-MCPD caused mitochondrial damage in renal cells by inhibiting the SIRT3/SOD2 pathway. Further, we found that 3-MCPD could interfere with rhythm protein BMAL1 expression at protein and mRNA levels in mice kidney and NRK-52E cells. Simultaneously, the balance of the daily oscillation of SIRT3/SOD2 pathway proteins was impeded under 3-MCPD treatment. To determine the role of BAML1 in mitochondrial damage, we overexpressed the BMAL1 protein. The data showed that BMAL1 overexpression upregulated SIRT3 and SOD2 expression and attenuated mitochondrial damage caused by 3-MCPD. These results indicated that 3-MCPD inhibited the SIRT3/SOD2 pathway by affecting the expression of the rhythm protein BMAL1, thereby inducing mitochondrial damage in renal cells. Taken together, our work reveals that 3-MCPD may possess a toxic effect via circadian clock mechanisms.

Activating autophagy and ferroptosis of 3­Chloropropane­1,2­diol induces injury of human umbilical vein endothelial cells via AMPK/mTOR/ULK1.

3­Chloropropane­1,2­diol (3­MCPD) is an internationally recognized food pollutant. 3­MCPD has reproductive, renal and neurotoxic properties. However, whether 3­MCPD induces human umbilical vein endothelial cell (HUVEC) injury has not been previously reported. In the present study, HUVECs were treated using 2 µg/ml 3­MCPD for 24 h at 37˚C. The effects of 3­MCPD on HUVEC proliferation and cell cycle arrest, death and senescence were then assessed using Cell Counting Kit­8 (CCK­8), flow cytometry and ß­galactosidase staining, respectively. Whether 3­MCPD induced ferroptosis was evaluated using JC­1 and FerroOrange staining and transmission electron microscopy. A small interfering RNA targeting AMPK was used to assess whether 3­MCPD promoted ferroptosis via AMPK signaling. The results demonstrated that 3­MCPD inhibited HUVEC proliferation in a dose­dependent manner and induced cell cycle arrest. Furthermore, 3­MCPD promoted senescence in HUVECs with elevated DNA damage and cell death. The CCK­8 results demonstrated that ferroptosis and autophagy inhibitors significantly reversed cell death caused by 3­MCPD. Moreover, 3­MCPD increased mitochondrial membrane potential, which indicated that 3­MCPD contributed to mitochondrial dysfunction. 3­MCPD also markedly increased intracellular Fe2+ levels and lipid peroxidation in HUVECs. The present study assessed the underlying mechanism by which 3­MCPD activated autophagy and ferroptosis in HUVECs. The data demonstrated that 3­MCPD significantly increased phosphorylation levels of AMPK and unc­51 like autophagy activating kinase (ULK1) but significantly decreased phosphorylation of mTOR in HUVECs. Furthermore, silencing of AMPK significantly reversed the increase in autophagy, lipid peroxidation and Fe2+ induced by 3­MCPD. In conclusion, 3­MCPD demonstrated a significant damaging effect on HUVECs via induction of autophagy and ferroptosis; such effects may be mediated by AMPK/mTOR/ULK1 signaling. To the best of our knowledge, the present study was the first to demonstrate the mechanism of 3­MCPD­induced vascular endothelial cell injury and lays a molecular foundation for the prevention of 3­MCPD­related vascular diseases.

The role of PGC-1α and metabolic signaling pathway in kidney injury following chronic administration with 3-MCPD as a food processing contaminant.

3-Monochloropropane-1,2-diol (3-MCPD) as a byproduct of food processing and a carcinogenic agent has attracted much attention in the last decades. Kidney is the main target organ that is sensitive to the toxicity of 3-MCPD. Due to limited evidence about possible 3-MCPD toxicity, we design an investigation to determine the role of mitochondrial biogenesis following chronic oral administration of 3-MCPD (2, 4, 8 and 32 mg/kg) for 2 months in male C57 mice. The present study evaluated the affects of 3-MCPD in modulating metabolic signalling which is associated with Il-18, PGC-1α, Nrf-2 and Sir3 which are the major transcription factors. Our data confirms controversial behaviors after chronic exposure with 3-MCPD. Over expression of the PGC-1α and Sir3 and IL-18 were observed after exposure with 2,4 & 8 mg kg-1  day-1 of 3-MCPD. In front, PGC-1α down-regulation occurs at the highest dose (32 mg/kg) resulted in kidney injury. Based on the findings, PGC-1α plays an important role in the restoration of the mitochondrial function during the recovery from chronic kidney injury. We suggest that the PGC-1α can be consider as a therapeutic target in prevention and treatment of kidney injury after chronic exposure of 3-MCPD. PRACTICAL APPLICATIONS: 3-Monochloropropane-1, 2-diol (3-MCPD) existed in several foods, can induce nephrotoxicity, progressive nephropathy and renal tubule dilation following acute and chronic exposure. It revealed that 3-MCPD toxicity is related to metabolites which can cause oxidative stress and activation of cell death signaling. It seems that cytotoxicity of 3-MCPD has disruptive effect on kidney cells due to rise in ROS production and decrease in mitochondrial membrane permeability. These effects can lead to MPT pore opening, cytochrome c release and activation of programed cell death signaling pathway. Therefore, present study was investigated the role of PGC-1a and the metabolic signaling involved in 3-MCPD-induced nephrotoxicity for the first time. Our data revealed that up-regulation of mitochondrial biogenesis following chronic exposure with 3-MCPD accelerates recovery of mitochondrial and cellular function in kidney by deacetylation of histones, overexpression of transcription factors (PGC-1α, Nrf-2, and Sir3) and maintaining cellular homeostasis.

Metabolites of 2- and 3-Monochloropropanediol (2- and 3-MCPD) in Humans: Urinary Excretion of 2-Chlorohydracrylic Acid and 3-Chlorolactic Acid after Controlled Exposure to a Single High Dose of Fatty Acid Esters of 2- and 3-MCPD.

SCOPE: Fatty acid esters of 2-monochloropropane-1,3-diol (2-MCPD) and 3-monochloropropane-1,2-diol (3-MCPD) are formed during the deodorization of vegetable oils. After lipase-catalyzed hydrolysis in the intestine, 2- and 3-MCPD are absorbed, but their ensuing human metabolism is unknown. METHODS AND RESULTS: The compounds 2-chlorohydracrylic acid (2-ClHA) and 3-chlorolactic acid (3-ClLA) resulting from oxidative metabolism of 2-MCPD and 3-MCPD, respectively, are identified and quantified in human urine samples. An exposure study with 12 adults is conducted to determine the urinary excretion of 2-ClHA and 3-ClLA. The participants eat 12 g of hazelnut oil containing 24.2 mg kg-1 2-MCPD and 54.5 mg kg-1 3-MCPD in the form of fatty acid esters. Average daily amounts of "background" excretion before the exposure are 69 nmol 2-ClHA and 3.0 nmol 3-ClLA. The additional mean excretion due to the uptake of the hazelnut oil amounts to 893 nmol 2-ClHA (34.0% of the 2-MCPD dose) and 16.4 nmol 3-ClLA (0.28% of the 3-MPCD dose). CONCLUSIONS: The products of oxidative metabolism of 2- and 3-MCPD, 2-ClHA, and 3-ClLA, are described for the first time in humans. Due to the lack of specificity, the metabolites may not be used as exposure biomarkers to low doses of bound 2- and 3-MCPD, respectively.

Assessment of intestinal absorption/metabolism of 3-chloro-1,2-propanediol (3-MCPD) and three 3-MCPD monoesters by Caco-2 cells.

3-chloro-1,2-propanediol (3-MCPD) and 3-MCPD esters are contaminants present in a variety of processed foods, including infant formulas. Toxicological data are unavailable in humans, but rodent studies have demonstrated renal and testicular toxicity from 3-MCPD and 3-MCPD esters. There is evidence that 3-MCPD esters are hydrolyzed in the digestive system, releasing 3-MCPD that would be absorbed and induce damage. We assessed absorption and metabolism of 3-MCPD and three 3-MCPD monoesters, 1-oleoyl (1-Ol), 1-linoleoyl (1-Li) and 1-palmitoyl (1-Pa) commonly found in U.S. infant formula using differentiated Caco-2 cells. After 1-hour incubation, all three monoesters released free 3-MCPD and free fatty acids (FFA) into Caco-2 cell supernatants. Free 3-MCPD had a high apparent permeability (Papp = 30.36 ± 1.31 cm/s × 10-6) suggesting that it is freely diffusible and highly absorbed by intestinal epithelium. 1-Li released 3-4-fold more 3-MCPD than 1-Ol and 1-Pa over 1 h, suggesting that this variable release rates might contribute to the overall in vivo exposure to 3-MCPD. None of the monoesters or FFA were detected in basolateral supernatants, suggesting that these compounds do not cross the intestinal wall without further transformation. In summary, this study provides relevant data to advance knowledge of in vivo intestinal absorption and metabolism of 3-MCPD monoesters.

Ninety-Day Nephrotoxicity Evaluation of 3-MCPD 1-Monooleate and 1-Monostearate Exposures in Male Sprague Dawley Rats Using Proteomic Analysis.

Fatty acid esters of 3-monochloropropane 1,2-diol (3-MCPD esters) are processing-induced food toxicants, with the kidney as their major target organ. For the first time, this study treated Sprague Dawley (SD) rats with 3-MCPD 1-monooleate at 10 and 100 mg/kg BW/day and 1-monostearate at 15 and 150 mg/kg BW/day for 90 days and examined for their potential semi-long-term nephrotoxicity and the associated molecular mechanisms. No bodyweight difference was observed between groups during the study. Both 3-MCPD 1-monooleate and 1-monostearate resulted in a dose-dependent increase of serum urea creatinine, uric acid and urea nitrogen levels, and histological renal impairment. The proteomic analysis of the kidney samples showed that the 3-MCPD esters deregulated proteins involved in the pathways for ion transportation, apoptosis, the metabolism of xenobiotics, and enzymes related to endogenous biological metabolisms of carbohydrates, amino acids, nitrogen, lipids, fatty acids, and the tricarboxylic acid (TCA) cycle, providing partial explanation for the nephrotoxicity of 3-MCPD esters.

Molecular modification of a halohydrin dehalogenase for kinetic regulation to synthesize optically pure (S)-epichlorohydrin.

Asymmetric synthesis of chiral epichlorohydrin (ECH) from 1,3-dichloro-2-propanol (1,3-DCP) using halohydrin dehalogenases (HHDHs) is of great value due to the 100% theoretical yield and high enantioselectivity. The vital problem in the asymmetric synthesis is to prepare optically pure ECH. In this study, key amino acid residues located at halide ion channels of HheC (P175S/W249P) (HheCPS) were modified to regulate the kinetic parameters. HheCPS I81W, F86N and V94R were constructed with the corresponding halide ion channels destroyed. The catalytically efficiencies (kcat/Km) of the three mutants exhibited 0.38-, 0.23- and 0.23-fold decrease toward (S)-ECH and the reverse reaction was significantly inhibited. As the results, (S)-ECH was synthesized with >99% enantiomeric excess (e.e.) and 63.42%, 67.08% and 57.01% yields, respectively, under 20 mM 1,3-DCP as substrate. To our knowledge, this is the first investigation of the molecule kinetic modification of HHDHs and also the first report for the biosynthesis of optically pure (S)-ECH from 1,3-DCP using HHDHs.

Metabolomics-based biomarker analysis of dihydroxypropyl mercapturic acid isomers from 3-monochloropropane-1,2-diol and glycidol for evaluation of toxicokinetics in rats and daily internal exposure in humans.

3-Monochloropropane-1,2-diol (3-MCPD), glycidol, and their esters are some major sources of risk factors during food processing. Here we showed the biomarker analysis of 2,3-dihydroxypropyl mercapturic acid (DHPMA) isomers which derived from the metabolism of 3-MCPD, glycidol, and their esters in urine of rats and humans. Iso-DHPMA, a novel urinary metabolite, was discovered and detected in urine of rats, which were orally administered with glycidol but not 3-MCPD. Using the quadrupole-orbitrap high-resolution mass spectrometry, we confirmed that iso-DHPMA appeared a specific biomarker which derived from glycidol. The limit of quantification (signal-to-noise ratio, 10:1) of the analytes in urine of rats and humans were 1.33 ng/mL and 1.56 ng/mL, respectively. Acceptable within-laboratory reproducibility (RSD<9.0%) and spiking recovery (94.7%-100.1%) substantially supported the use of current method for robust biomarker analysis, which was successfully applied to the toxicokinetic study of DHPMA in rats and short-term internal exposure to 3-MCPD and glycidol in humans.

Covalent immobilization of halohydrin dehalogenase for efficient synthesis of epichlorohydrin in an integrated bioreactor.

Halohydrin dehalogenase (HHDH)-mediated dehalogenation of 1,3-dichloro-2-propanol (1,3-DCP) is a key step in the chemoenzymatic synthesis of epichlorohydrin (ECH) from glycerol. In this study, a covalent immobilization strategy was employed to enhance the stability of Agrobacterium tumefaciens HHDH using epoxy resin ES-103B as a carrier. Under optimal conditions, the activity recovery of ES-103B-immobilized HHDH (HHDH@ES-103B) was 62.4% and the specific activity was 1604 U/g. The HHDH@ES-103B exhibited excellent thermostability, with a half-life of 68.6 days at 40°C, which is 8.0-times higher than that of the free HHDH. A semicontinuous biotransformation of 1,3-DCP to ECH was performed using HHDH@ES-103B as biocatalyst in a recirculating packed bed reactor (RPBR), resulting in an ECH yield of 94.2%, with an average productivity of 5.2 g/L/h. The RPBR system exhibited a high operational stability and even after 50 cycles of reaction, it retained > 90% of the initial conversion. Furthermore, an integrated bioprocess based on in situ product recovery (ISPR) was developed in RPBR to overcome product inhibition. The integrated bioreactor equipped with an external macroporous adsorption resin HZD-9 column led to another 1.6-fold increase in ECH productivity to 8.46 g/L/h. This improved stability and reusability of HHDH@ES-103B demonstrated its potential for the biotransformation of 1,3-DCP to ECH. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:784-792, 2018.

Toxicokinetics and Metabolism of 3-Monochloropropane 1,2-Diol Dipalmitate in Sprague Dawley Rats.

Fatty acid esters of 3-monochloropropane 1,2-diol (3-MCPD) are a group of processing-induced toxicants. To better clarify their possible toxicological effects and mechanisms, it is important to investigate their absorption, distribution, metabolism, and excretion. In this study, the kinetic parameters of 3-MCPD dipalmitate in Sprague Dawley (SD) rat plasma were determined using ultraperformance liquid chromatography-triple quadrupole mass spectrometry. 3-MCPD dipalmitate was absorbed in rats with a Cmax of 135.00 ng/mL, a T1/2 of 3.87 h, a Tmax of 2.5 h, an MRT of 5.08 h, a CL of 3.50 L/h/g, a Vd of 21.34 L/g, and an AUC0-∞ of 458.47 h·ng/mL. A total of 17 metabolites were identified, and 16 of them were reported for the first time. Furthermore, these metabolites were examined for their presences in the liver, kidney, testis, brain, spleen, thymus, intestine, plasma, feces, and urine samples 2, 6, 12, 24, and 48 h after oral administration of 3-MCPD dipalmitate using Metabolynx software.

Effects of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites on DNA damage and repair under in vitro conditions.

3-monochloropropane-1,2-diol (3-MCPD) is a food contaminant that occurs during industrial production processes and can be found mainly in fat and salt containing products. 3-MCPD has exhibited mutagenic activity in vitro but not in vivo, however, a genotoxic mechanism for the occurrence of kidney tumors has not so far been excluded. The main pathway of mammalian 3-MCPD metabolism is via the formation of ß--chlorolactatic acid and formation of glycidol has been demonstrated in bacterial metabolism. The aim of this study was to investigate genotoxic and oxidative DNA damaging effects of 3-MCPD and its metabolites, and to provide a better understanding of their roles in DNA repair processes. DNA damage was assessed by alkaline comet assay in target rat kidney epithelial cell lines (NRK-52E) and human embryonic kidney cells (HEK-293). Purine and pyrimidine base damage, H2O2 sensitivity and DNA repair capacity were assessed via modified comet assay. The results revealed in vitro evidence for increased genotoxicity and H2O2 sensitivity. No association was found between oxidative DNA damage and DNA repair capacity with the exception of glycidol treatment at 20 µg/mL. These findings provide further insights into the mechanisms underlying the in vitro genotoxic potential of 3-MCPD and metabolites.

Collaborative Study of an Indirect Enzymatic Method for the Simultaneous Analysis of 3-MCPD, 2-MCPD, and Glycidyl Esters in Edible Oils.

A collaborative study was conducted to evaluate an indirect enzymatic method for the analysis of fatty acid esters of 3-monochloro-1,2-propanediol (3-MCPD), 2-monochloro-1,3-propanediol (2-MCPD), and glycidol (Gly) in edible oils and fats. The method is characterized by the use of Candida rugosa lipase, which hydrolyzes the esters at room temperature in 30 min. Hydrolysis and bromination steps convert esters of 3-MCPD, 2-MCPD, and glycidol to free 3-MCPD, 2-MCPD, and 3-monobromo-1,2-propanediol, respectively, which are then derivatized with phenylboronic acid, and analyzed by gas chromatography-mass spectrometry. In a collaborative study involving 13 laboratories, liquid palm, solid palm, rapeseed, and rice bran oils spiked with 0.5-4.4 mg/kg of esters of 3-MCPD, 2-MCPD, and Gly were analyzed in duplicate. The repeatability (RSDr) were < 5% for five liquid oil samples and 8% for a solid oil sample. The reproducibility (RSDR) ranged from 5% to 18% for all oil samples. These RSDR values were considered satisfactory because the Horwitz ratios were ≤ 1.3% for all three analytes in all oil samples. This method is applicable to the quantification of 3-MCPD, 2-MCPD, and Gly esters in edible oils.

Involvement of cytochrome P4502E1 in the toxicity of dichloropropanol to rat hepatocyte cultures.

Hepatocytes were isolated and cultured from untreated rats and rats treated with isoniazid to induce cytochrome P4502E1. Isoniazid selectively increased p-nitrophenol hydroxylase activity in 2-h cultures, and increased the toxicity of both 1,3- and 2,3-dichloropropanol. Isoniazid also increased the rate and extent of glutathione depletion by the dichloropropanols. There was no effect of isoniazid on the toxicity of 1,3-dichloroacetone, precocene II or allyl alcohol. In addition, diethyldithiocarbamate selectively inhibited p-nitrophenol hydroxylase in 2-h cultures from untreated and isoniazid-treated rats, as well as abolishing toxicity of the dichloropropanols. In 24-h cultures from isoniazid-treated rats diethyldithiocarbamate inhibited high affinity MCOD activity by 55% and there was also a small but significant inhibition of precocene II toxicity. These results indicate that isoniazid-inducible P4502E1 can mediate the toxicity of dichloropropanol.