Chloroacetaldehyde Research Articles

An on-line SPE-LC-MS/MS method for quantification of nucleobases and nucleosides present in biological fluids.

Solid phase extraction (SPE) and liquid chromatographic (LC) separation of nucleobases and nucleosides are challenging due to the high hydrophilicity of these compounds. Herein we report a novel on-line SPE-LC-MS/MS method for their quantification after pre-column derivatization with chloroacetaldehyde (CAA). The method proposed is selective and sensitive with limits of detection at the nano-molar level. Analysis of urine and saliva samples by using this method is demonstrated. Adenine, guanine, cytosine, adenosine, guanosine, and cytidine were found in the range from 0.19 (guanosine) to 1.83 μM (cytidine) in urine and from 0.015 (guanosine) to 0.79 μM (adenine) in saliva. Interestingly, methylation of cytidine was found to be significantly different in urine from that in saliva. While 5-hydroxymethylcytidine was detected at a very low level (<0.05 μM) in saliva, it was found to be the most prominent methylated cytidine in urine at a high level of 3.33 μM. Since on-line SPE is deployed, the proposed LC-MS/MS quantitative assay is convenient to carry out and offers good assay accuracy and repeatability.

Synthesis of [Ta6O19] @MOF composite as a chloroacetaldehyde optical sensor in wastewater obtained from two waste-resourced precursors: Tantalum from capacitors and terephthalic acid from PET bottle

Metal-organic frameworks (MOFs) can be derived from waste and used for various purposes to address environmental issues. MOFs possess superior adsorption and regeneration capabilities, making them effective for capturing various pollutants found in wastewater. Here, a rare Lindqvist polyoxometalate (POM), (Na8[Ta6O19]), was synthesized and used to fabricate a novel MOF-based composite (POTa@UiO-66) through a one-pot reaction with zirconium chloride (ZrCl4) and terephthalic acid (TPA). Some building blocks of the composite was then prepared using recycled materials obtained from capacitors and plastic bottles. According to FT-IR, SEM, UV–visible, and fluorescence analysis results, both composites showed structural similarity and exhibited efficient fluorescence detection properties for chloroacetaldehyde (CA) through the common aldehydes addition in aqueous media (at λex = 300 nm). A dynamic quenching process was suggested between the sensor and CA based on absorption spectra and Stern-Volmer plots. The low detection limits for both composites prepared by pure and recycled precursors were 5.3 × 10−8 M and 1.5 × 10−7 M, respectively.

A novel one-pot fluorescence tagging and depyrimidination strategy for quantification of global DNA methylation

DNA methylation is intensively studied in medical science. Current HPLC methods for quantification of global DNA methylation involve digestion of a DNA sample and HPLC determination of both cytosine (C) and 5-methylcytosine (5mC) so that percentage of 5mC in total cytosine can be calculated as DNA methylation level. Herein we report a novel HPLC method based on a one-pot fluorescence tagging and depyrimidination reaction between DNA and chloroacetaldehyde (CAA) for highly sensitive quantification of global DNA methylation. In the one-pot reaction, C and 5mC residues in a DNA sequence react with CAA, forming fluorescent etheno-adducts that are then released from the sequence through depyrimidination. Interestingly, etheno-5mC (ε-5mC) is ∼20 times more fluorescent than ε-C and other ε-nucleobases resulting from the reaction, which greatly facilitates the quantification. Further, due to the tagging-induced increase in structural aromaticity, ε-nucleobases are far more separable by HPLC than intact nucleobases. The proposed HPLC method with fluorescence detection (HPLC-FD) is quick (i.e., < 1h per assay) and highly sensitive with a detection limit of 0.80 nM (or 250 fg on column) for 5mC. Using the method, DNA samples isolated from yeast, HCT-116 cells, and tissues were analyzed. Global DNA methylation was measured to be in the range from 0.35% to 2.23% in the samples analyzed. This sensitive method allowed accurate analyses of minute DNA samples (∼100 ng) isolated from milligrams of tissues.

Kinetics and mechanism of haloacetaldehyde formation from the reaction of acetaldehyde and chlorine

Haloacetaldehydes (HALs) are the third prevalent group of disinfection by-products (DBPs) by weight in drinking water, and their cytotoxicity and genotoxicity are higher than regulated DBPs. In order to understand their formation mechanism during chlorination and ozonation-chlorination, this study examined the reaction kinetics of chloral hydrate (CH), dichloroacetaldehyde (DCA), chloroacetaldehyde (CA) and acetaldehyde by chlorine at different pH values and chlorine doses. The results showed that the reaction rate constants increased with pH and chlorine dose, except that the degradation of CH would not be affected by the presence of free chlorine. At the same pH and chlorine dose, the half-lives of CH, DCA, CA and acetaldehyde were in the order of CH > acetaldehyde ≫ DCA > CA. A kinetic model used to predict the formation of HALs and chloroform during chlorination of acetaldehyde was developed, and the predicted data fitted well with the measured data. As pre-ozonation could oxidize natural organic matter to acetaldehydes, the concentration of acetaldehyde formed after pre-ozonation was used to calculate the HAL yields during ozonation-chlorination by the kinetic model, which fitted the experimental results well. The kinetic model elucidated that the formation mechanism of HALs was a stepwise substitution process on the α-hydrogen of acetaldehyde during chlorination.

Protection by ginseng saponins against cyclophosphamide-induced liver injuries in rats by induction of cytochrome P450 expression and mediation of the l-arginine/nitric oxide pathway based on metabolomics.

Ginseng saponins (GS) are the main active compounds in Panax ginseng and have been proven to be highly effective in attenuating the side effects of chemotherapy. However, there have been no reports on the mechanism of action of GS. Treatment with GS has certain benefits, including decreasing the toxicity levels in the liver [alanine aminotransferase (ALT), albumin (ALB), alkaline phosphatase (ALP), aspartate transaminase (AST)], reducing oxidative stress [malondialdehyde (MDA), nitric oxide (NO)], diminishing inflammatory factors [interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels], and augmenting the levels of glutathione (GSH) and superoxide dismutase (SOD). The pharmacokinetics study showed that the area under the curve from 0 to 24 hr (AUC 0-24 hr) of 4-ketocyclophosphamide (4-KetoCTX) and carboxyphosphamide (CPM) was significantly increased after GS treatment. This study found that GS treatment can reduce chloroacetaldehyde (CAA) production by affecting CYP3A4, CYP2B6, and CYP2C9 protein expression in the liver. For the metabolomics study, GS attenuated the abnormalities of amino acid metabolic pathways in CP-induced liver injuries of rats and significantly enhanced the l-arginine level while reducing the serum nitric oxide (NO) level. This outcome was confirmed by the inhibition of the activities of NO synthase in the liver of rats.

Rational design to enhance the catalytic activity of 2-deoxy-D-ribose-5-phosphate aldolase from Pseudomonas syringae pv. syringae B728a

The 2-Deoxy-d-ribose-5-phosphate aldolase (DERA) enzyme in psychrophilic bacteria has gradually attracted the attention of researchers. A novel gene, deoC (681 bp), encoding DERAPsy, was identified in Pseudomonas syringae pv. syringae B728a, recombinantly expressed in E. coli BL21 and purified via affinity chromatography, which yielded a homodimeric enzyme of 23 kDa. The specific activity of DERAPsy toward 2-deoxy-d-ribose-5-phosphate (DR5P) was 7.37 ± 0.03 U/mg, and 61.32% of its initial activity remained after incubation in 300 mM acetaldehyde at 25 °C for 2 h. Based on the calculation results (dock binding free energy) with the ligand chloroacetaldehyde (CAH), five target substitutions (T16L, F69R, V66K, S188V, and G189R) were identified, in which the DERAPsy mutant (G189R) exhibited higher catalytic activity toward DR5P than DERAPsy. Only the DERAPsy mutant (V66K) exhibited 12% higher activity toward chloroacetaldehyde and acetaldehyde condensation reactions than DERAPsy. Fortunately, the aldehyde tolerance of these mutants exhibited no significant decline compared with the wild type. These results indicate an effective strategy for enhancing DERA activity.

Insights into chloroacetaldehydes degradation by 254 nm ultraviolet: Kinetics, products, and influencing factors

Haloacetaldehydes (HALs) are a type of disinfection byproducts (DBPs) frequently found in disinfected drinking water, and their effective control is important to safeguard human health. To evaluate the appropriateness of using ultraviolet technology for drinking water treatment, this study comparatively examines the photolysis of three chlorinated HALs (Cl-HALs), including monochloroacetaldehyde (MCAL), dichloroacetaldehyde (DCAL), and trichloroacetaldehyde (TCAL), under varying operational and environmental conditions by 254 nm ultraviolet (UV254). Results show that they can be degraded within 2 h at an initial concentration of 20 μg/L with pseudo-first-order reaction rates ranked as MCAL > DCAL > TCAL. Chloride was the major product and total organic carbon decreased substantially, indicating that UV irradiation of Cl-HALs mainly underwent mineralization. MCAL was not formed during photolysis of DCAL or TCAL, suggesting that the carbon-chloride bonds in Cl-HALs were cleaved simultaneously rather than sequentially. In terms of influencing factors, adding free chlorine into solution apparently enhanced Cl-HALs photolysis, whereas increasing Cl-HALs dosage, nitrate, bromide, or iodide inhibited Cl-HAL photolysis. Under their combined effects, the photolysis rates of Cl-HALs dosed into tap and swimming pool waters were lower than those dosed into ultrapure water. These findings hence provide insights for better understanding the treatability of UV254 for Cl-HALs control.

Novel insights into the mechanism of cyclophosphamide-induced bladder toxicity: chloroacetaldehyde's contribution to urothelial dysfunction in vitro.

The clinical use of cyclophosphamide and ifosfamide is limited by a resultant bladder toxicity which has been attributed to the metabolite acrolein. Another metabolite chloroacetaldehyde (CAA) associated with nephrotoxicity, has not been investigated for toxicity in the bladder and this study investigates the effects of acrolein and CAA on human urothelial cells in vitro. Human urothelial cells (RT4 and T24) were treated with acrolein or CAA and changes in cell viability, reactive oxygen species, caspase-3 activity and release of urothelial mediators ATP, acetylcholine, PGE2 were measured. The protective effects of N-acetyl cysteine (NAC) were also assessed. Both metabolites were toxic to human urothelial cells, however, CAA significantly decreased cell viability at a ten-fold lower concentration (10µM) than acrolein (100µM). This was associated with increased ROS production and caspase-3 activity. NAC protected cells from these changes. In RT4 cells 100µM acrolein caused a significant increase in basal and stretch-induced ATP, Ach and PGE2 release. In T24 cells chloroacetaldehyde (10µM) increased basal and stimulated ATP and PGE2 levels. Again, NAC protected against changes in urothelial mediator release following acrolein or CAA. This study is the first to report that CAA in addition to acrolein contributes to the urotoxicity of cyclophosphamide and ifosfamide. Both metabolites altered urothelial mediator levels which could contribute to the sensory and functional bladder changes experienced by patients after treatment with cyclophosphamide or ifosfamide. Alterations in urothelial cell viability and mediator release may be causally linked to oxidative stress, with NAC providing protection against these changes.

Wuzhi capsule regulates chloroacetaldehyde pharmacokinetics behaviour and alleviates high-dose cyclophosphamide-induced nephrotoxicity and neurotoxicity in rats.

High-dose cyclophosphamide (HD-CTX) treatment often leads to severe nephrotoxicity and neurotoxicity, which are mainly caused by one of its metabolites, chloroacetaldehyde (CAA). However, there are no effective antidotes to prevent these side effects. The objective of this study was to evaluate the effect of Wuzhi Capsule (WZC) on the pharmacokinetics of CTX and its metabolites in rats, and the attenuation of CAA induced kidney and brain injuries, which was produced at equimolar with 2-dechloroethylcyclophosphamide. Rats were treated with single- or multiple-dose of WZC when giving HD-CTX, and the plasma concentration of CTX and its metabolites were quantitated by UHPLC-MS/MS Single-dose, not multiple-dose of WZC co-administration (300mg/kg) significantly reduced Cmax and AUC0→24h of DC-CTX by 33.10% and 35.51%, respectively. Biochemical assay suggested oxidative stress was involved in kidney and brain injuries by HD-CTX, which were attenuated by single-dose WZC (300mg/kg) pre-treatment, with increased glutathione, glutathione peroxidase and superoxide dismutase contents/or activities in both tissues and plasma (P<0.05). Meanwhile, WZC pre-treatment could also significantly decrease the plasma levels of creatinine, blood urea nitrogen and malondialdehyde (P<0.05). Additionally, WZC treatment improved the morphology and pathology condition of the kidneys and brains in rats. In conclusion, single-dose WZC co-administration decreased CAA production and exerted protective effect on CTX-induced oxidative stress in kidney and brain, whereas repetitive WZC co-administration with CTX was probably not recommended.

The response of Escherichia coli to the alkylating agents chloroacetaldehyde and styrene oxide

DNA damage is ubiquitous and can arise from endogenous or exogenous sources. DNA-damaging alkylating agents are present in environmental toxicants as well as in cancer chemotherapy drugs and are a constant threat, which can lead to mutations or cell death. All organisms have multiple DNA repair and DNA damage tolerance pathways to resist the potentially negative effects of exposure to alkylating agents. In bacteria, many of the genes in these pathways are regulated as part of the SOS reponse or the adaptive response. In this work, we probed the cellular responses to the alkylating agents chloroacetaldehyde (CAA), which is a metabolite of 1,2-dichloroethane used to produce polyvinyl chloride, and styrene oxide (SO), a major metabolite of styrene used in the production of polystyrene and other polymers. Vinyl chloride and styrene are produced on an industrial scale of billions of kilograms annually and thus have a high potential for environmental exposure. To identify stress response genes in E. coli that are responsible for tolerance to the reactive metabolites CAA and SO, we used libraries of transcriptional reporters and gene deletion strains. In response to both alkylating agents, genes associated with several different stress pathways were upregulated, including protein, membrane, and oxidative stress, as well as DNA damage. E. coli strains lacking genes involved in base excision repair and nucleotide excision repair were sensitive to SO, whereas strains lacking recA and the SOS gene ybfE were sensitive to both alkylating agents tested. This work indicates the varied systems involved in cellular responses to alkylating agents, and highlights the specific DNA repair genes involved in the responses.

Effects of ketoconazole on cyclophosphamide metabolism: evaluation of CYP3A4 inhibition effect using the in vitro and in vivo models.

Cyclophosphamide (CP) is widely used in anticancer therapy regimens and 2-dechloroethylcyclophosphamide (DECP) is its side-chain dechloroethylated metabolite. N-dechloroethylation of CP mediated by the enzyme CYP3A4 yields nephrotoxic and neurotoxic chloroacetaldehyde (CAA) in equimolar amount to DECP. This study aimed to evaluate the inhibitory effect of ketoconazole (KTZ) on CP metabolism through in vitro and in vivo drug-drug interaction (DDI) research. Long-term treatment of KTZ induces hepatic injury; thus single doses of KTZ at low, middle, and high levels (10, 20, and 40 mg/kg) were investigated for pharmacokinetic DDI with CP. Our in vitro human liver microsome modeling approach suggested that KTZ inhibited CYP3A4 activity and then decreased DECP exposure. In addition, an UHPLC-MS/MS method for quantifying CP, DECP, and KTZ in rat plasma was developed and fully validated with a 4 min analysis coupled with a simple and reproducible one-step protein precipitation. A further in vivo pharmacokinetic study demonstrated that combination use of CP (10 mg/kg) and KTZ (10, 20, and 40 mg/kg) in rats caused a KTZ dose-dependent decrease in main parameters of DECP (Cmax, Tmax, and AUC0–∞) and provided magnitude exposure of DECP (more than a 50% AUC decrease) as a consequence of CYP3A inhibition but had only a small effect on the CP plasma concentration. Our results suggested that combination usage of a CYP3A4 inhibitor like KTZ may decrease CAA exposure and thus intervene against CAA-induced adverse effects in CP clinical treatment.

Time- and NADPH-Dependent Inhibition on CYP3A by Gomisin A and the Pharmacokinetic Interactions between Gomisin A and Cyclophosphamide in Rats

The traditional Chinese medicine Schisandra chinensis has remarkable protective effects against chemical-induced toxicity. Cyclophosphamide (CTX), in spite advances in chemotherapy and immunosuppressive regimes, is prone to cause severe toxicity due to its chloroacetaldehyde (CAA) metabolite produced by CYP3A. Our previous study identified that S. chinensis extract (SCE) co-administration potently decreased CAA production and attenuated liver, kidney and brain injuries in CTX-treated rats. Gomisin A (Gom A) is proved to be one of the most abundant bioactive lignans in S. chinensis with a significant CYP3A inhibitory effect. To find out whether and how Gom A participated in the chemoprevention of SCE against CTX toxicity, the Gom A-caused CYP3A inhibition in vitro as well as the pharmacokinetic interactions between Gom A and CTX in vivo were examined in this study. Using human liver microsomes, a reversible inhibition assay revealed that Gom A was a competitive inhibitor with a KI value of 1.10 µM, and the time- and NADPH-dependent CYP3A inhibition of Gom A was observed in a time-dependent inhibition assay (KI = 0.35 µM, kinact = 1.96 min−1). Hepatic CYP3A mRNA expression experienced a significant increase in our rat model with Gom A administration. This explained why CAA production decreased in the 0.5 h- and 6 h-pretreatment rat groups while it increased in the 24 h- and 72 h-pretreatment groups, indicating a bidirectional effect of Gom A on CYP3A-mediated CTX metabolism. The present study suggested that Gom A participates like SCE in the pharmacokinetic intervention of CTX by blocking CYP3A-mediated metabolism and reducing CAA production, and thus plays an important role in the chemopreventive activity of S. chinensis against CTX toxicity, in addition to the previously recognized protective effects. Also, the combined use of S. chinensis preparation or other drugs containing Gom A as the main component with CTX needed to be addressed for better clinical intervention.

Determination of Escherichia coli Genes Important for DNA Repair Following Alkylation

Alkylating agents, common in industrial manufacturing and chemotherapy, can damage DNA by adding alkyl groups to nucleotides. Alkylation can decrease the stability of the N‐glycosidic sugar‐base bond, making it more apt to break; alkylation can also interfere with base pairing. Either of these perturbations can lead to mutations if not remedied. DNA repair mechanisms, including direct base repair, base excision repair, and nucleotide excision repair, play important roles in correcting these lesions. Our research aims to better understand the mechanisms behind repair of alkylated DNA, first by confirming which genes are most critical. An assay was designed to screen bacterial strains harboring single deletions of more than 20 genes, selected based on prior known association to DNA repair. The survival of E. coli AB1157 strains, each with one of the 20 genes deleted, was measured upon exposure to one of two alkylating agents: styrene oxide (SO) or chloroacetaldehyde (CAA). Strains with dam deleted were sensitive to SO and CAA, suggesting that DNA adenine methylase, the protein encoded by dam, plays a critical role in DNA alkylation repair. Strains lacking recA, which plays multiple roles in DNA damage responses, were quite sensitive to both agents. Strains with mfd, mutH, or mutY deleted were not more sensitive to SO and CAA than the wild‐type strains. Next, the survival of double knockouts will be assayed, which will help determine if these genes act in the same or different DNA repair pathways.Support or Funding InformationThis work was supported by the American Cancer Society and the NU Office of the Provost.

Vinyl Chloride Metabolites Potentiate Inflammatory Liver Injury Caused by LPS in Mice.

Vinyl chloride (VC) is a ubiquitous environmental contaminant for which human risk is incompletely understood. We have previously reported that high occupational exposure to VC directly caused liver damage in humans. However, whether VC may also potentiate liver injury from other causes is not known. C57Bl/6J mice were administered chloroethanol (CE), a major metabolite of VC, and lipopolysaccharide (LPS) 24 h after CE. Samples were harvested for determination of liver damage, inflammation, and changes in carbohydrate and lipid metabolism. In mice, CE exposure alone caused no detectable liver damage. LPS exposure caused inflammatory liver damage, oxidative stress, lipid accumulation, and glycogen depletion; the effect of all of these variables was potentiated by CE pre-exposure. In vitro experiments suggest that VC metabolite chloroacetaldehyde (CAA) directly damages mitochondria, which may explain the sensitization effect observed in vivo Moreover, co-exposure of cells to CAA and TNFα caused increased cell death, supporting the hypothesis of sensitization by VC metabolites. Taken together, these data demonstrate that exposure to VC/metabolites at levels that are not overtly hepatotoxic can potentiate liver injury caused by another hepatotoxicant. This serves as proof-of-concept that VC hepatotoxicity may be modified by an additional metabolic stress such as endotoxemia, which commonly occurs in acute (eg, sepsis) and chronic (eg, NAFLD) diseases.

Synthesis, Characterization, Docking and Biological Evaluation of Tetra Hydro Imidazo [1, 2-a] Pyrazine Derivatives

Novel Tetra hydro imidazo[1, 2-a] pyrazine derivatives have been synthesized by Treatment of pyrazine-2carboxylic acid with thionyl chloride in methanol yielded methyl pyrazine-2-carboxylate. This up on treatment with alcoholic ammonia gave pyrazinamide, further it was undergone Hoffman's degradation, gave 2-amino pyrazine after that it was refluxed for 26 hrs with chloro acetaldehyde, sodium bicarbonate gave imidazo[1, 2-a] pyrazine and hydrogen gas was passed on it in the presence of palladium on carbon yielded tetra hydro imidazo[1, 2-a] pyrazine, which was treated with different substituent's to get the novel derivatives. Synthesized compounds were identified and characterized by Melting point, TLC, FT-IR, 1H and 13C NMR, MASS and percent purity done by HPLC. Based on characterization and percent purity novel derivatives were subjected for molecular docking, QSAR studies and pharmacological evaluation. Selected compounds were docked on Anti oxidant activity using “Thioredoxin peroxidase B” as a target site. QSAR parameters like partition coefficient was studied and showed polar surface area below 140 A0 and passed ADME parameters. Analysis of docking and QSAR studies suggested that remarkable inhibitory activity was exhibited by the derivatives. Hydrogen bond interactions were mapped to confirm their potencies. They were evaluated for In-Vitro Anti-oxidant activity by DPPH method and In-Vitro Anti-diabetic activity by alpha-amylase inhibition assay by DNSA method. Results suggested that electron withdrawing groups like nitro, bromo having compounds shown potent activity. Rest of compounds showed mild to moderate activity.

Investigation of ifosfamide and chloroacetaldehyde renal toxicity through integration of in vitro liver-kidney microfluidic data and pharmacokinetic-system biology models.

We have integrated in vitro and in silico data to describe the toxicity of chloroacetaldehyde (CAA) on renal cells via its production from the metabolism of ifosfamide (IFO) by hepatic cells. A pharmacokinetic (PK) model described the production of CAA by the hepatocytes and its transport to the renal cells. A system biology model was coupled to the PK model to describe the production of reactive oxygen species (ROS) induced by CAA in the renal cells. In response to the ROS production, the metabolism of glutathione (GSH) and its depletion were modeled by the action of an NFE2L2 gene-dependent pathway. The model parameters were estimated in a Bayesian context via Markov Chain Monte Carlo (MCMC) simulations based on microfluidic experiments and literature in vitro data. Hepatic IFO and CAA in vitro intrinsic clearances were estimated to be 1.85 x 10(-9) μL s(-1) cell(-1) and 0.185 x 10(-9) μL s(-1) cell(-1) ,respectively (corresponding to an in vivo intrinsic IFO clearance estimate of 1.23 l h(-1) , to be compared to IFO published values ranging from 3 to 10 l h(-1) ). After model calibration, simulations made at therapeutic doses of IFO showed CAA renal intracellular concentrations ranging from 11 to 131 μM. Intracellular CAA concentrations above 70 μM induced intense ROS production and GSH depletion. Those responses were time and dose dependent, showing transient and non-linear kinetics. Those results are in agreement with literature data reporting that intracellular CAA toxic concentrations range from 35 to 320 μM, after therapeutic ifosfamide dosing. The results were also consistent with in vitro CAA renal cytotoxicity data.

Occurrence and Comparative Toxicity of Haloacetaldehyde Disinfection Byproducts in Drinking Water.

The introduction of drinking water disinfection greatly reduced waterborne diseases. However, the reaction between disinfectants and natural organic matter in the source water leads to an unintended consequence, the formation of drinking water disinfection byproducts (DBPs). The haloacetaldehydes (HALs) are the third largest group by weight of identified DBPs in drinking water. The primary objective of this study was to analyze the occurrence and comparative toxicity of the emerging HAL DBPs. A new HAL DBP, iodoacetaldehyde (IAL) was identified. This study provided the first systematic, quantitative comparison of HAL toxicity in Chinese hamster ovary cells. The rank order of HAL cytotoxicity is tribromoacetaldehyde (TBAL) ≈ chloroacetaldehyde (CAL) > dibromoacetaldehyde (DBAL) ≈ bromochloroacetaldehyde (BCAL) ≈ dibromochloroacetaldehyde (DBCAL) > IAL > bromoacetaldehyde (BAL) ≈ bromodichloroacetaldehyde (BDCAL) > dichloroacetaldehyde (DCAL) > trichloroacetaldehyde (TCAL). The HALs were highly cytotoxic compared to other DBP chemical classes. The rank order of HAL genotoxicity is DBAL > CAL ≈ DBCAL > TBAL ≈ BAL > BDCAL>BCAL ≈ DCAL>IAL. TCAL was not genotoxic. Because of their toxicity and abundance, further research is needed to investigate their mode of action to protect the public health and the environment.

The Nephrotoxic Ifosfamide-Metabolite Chloroacetaldehyde Interferes with Renal Extracellular Matrix Homeostasis

Background/Aims: Chronic renal proximal tubule dysfunction after therapy with the antineoplastic agent ifosfamide (IFO) is often attributed to the metabolite chloroacetaldehyde (CAA). Chronic IFO-nephropathy is reported to result in tubulointerstitial fibrosis and inflammation. Methods: To elucidate possible effects of CAA on extracellular matrix homeostasis, we investigated the action of CAA on markers of extracellular matrix (ECM) homeostasis in human proximal tubule cells (RPTEC) by use of direct ELISA for extracellular collagens and gelatin zymography. Results: An increase in type III collagen and a decrease in type IV collagen abundance in the media of RPTEC could be observed after exposure to CAA in clinically relevant concentrations. CAA increased intracellular type III and decreased intracellular type IV collagen. MMP-2 activity was decreased but MMP-9 activity unchanged. The enhanced CAA-induced collagen III formation could be attenuated by the intracellular Ca²⁺-chelator BAPTA-AM, the PKA-antagonist H-89 and by extracellular acidification. CAA-induced collagen III abundance was enhanced by db-cAMP and IBMX and by protein overload. Conclusions: CAA exerts profibrotic effects on RPTEC dependent on Ca²⁺ and cAMP/PKA-signaling. These effects are enhanced by additional protein burden and attenuated by acidification.

Classical oxazaphosphorines – metabolism and therapeutic properties – new implications

Cyclophosphamide (CPA) and ifosfamide (IFO) belong to oxazaphosphorine drugs and for a few decades have been widely used for treatment of solid tumours and haematological malignancies. Both drugs are administered in pharmacologically inactive form and require metabolic activation by cytochrome P-450 (CYP). Metabolic transformations taking place under the action of specific CYP isoenzymes lead to the formation of therapeutically essential metabolites and some toxic compounds affecting quality of therapy. The first stage of these conversions is connected with hydroxylation reactions occurring on the C-4 carbon atom within a ring and C-1 atoms of 2-chloroethyl chains. As a result of C-4 hydroxylation 4-hydroxy derivatives (4-OH-CPA and 4-OH-IFO) are formed and remain in tautomeric equilibrium with aldo compounds which in cancer cells spontaneously release cytotoxic phosphoramide mustards and urotoxic acrolein. At the same time hydroxychloroethyl compounds formed during hydroxylation of side-chains are unstable and collapse with the release of inter alia nephro- and neurotoxic chloroacetaldehyde (CAA). Due to formation of toxic metabolites it is essential to use some preventive agents such as mesna and recently examined agmatine. Since CPA and IFO are widely used anticancer drugs, their efficacy is limited not only by their toxicity but also due to occurring resistance. This resistance seems to be a result of changes of expression and activity of enzymes such as CYP and aldehyde dehydrogenase (ALDH) and increase of intracellular levels of glutathione (GSH) and glutathione S-transferase (GST). At present a few methods of overcoming this resistance are being examined including the use of metabolism modulators, antisense oligonucleotides selectively inhibiting gene expression, and introducing genes of some CYP isoenzymes to a cancer tissue.

Surface‐activated chemical ionization–electrospray mass spectrometry in the analysis of urinary thiodiglycolic acid

Thiodiglycolic acid (TDGA) is a urinary metabolite of the oxazaphosphorine class of chemotherapeutics, in particular of ifosfamide. Ifosfamide metabolism generates chloroacetaldehyde (CAA), a toxic compound associated with neurotoxicity, nephrotoxicity, urotoxicity and cardiotoxicity. CAA, in turn, interacts with cellular thiol groups leading to GSH depletion, cell death and generation of thiodiglycolic acid (TDGA), as a final product. TDGA is mainly excreted in the urine. The ability to accurately measure TDGA in urine, therefore, will be a useful way of monitoring exposure to ifosfamide during chemotherapy. TDGA in urine samples was measured with liquid chromatography coupled to mass spectrometry (LC/MS) by means of a novel Surface-Activated Chemical Ionization-Electrospray (SACI-ESI) or a classical ESI ion source alone. The SACI-ESI and ESI alone based methods for analysis of urinary TDGA were optimized and compared. A strong reduction in matrix effect together with enhanced quantification performances was obtained with the SACI-ESI when compared with ESI. In particular, an increase in quantification precision (from 85 to 95%) and accuracy (from 59 to 90%) were observed, which allowed for optimal detection of TDGA. The LC/SACI-ESI-MS approach provides a very sensitive and quantitative method for the analysis of TDGA. Thanks to the enhancement in sensitivity and matrix effect reduction, the SACI-ESI source enables the use of a relatively low-cost ion-trap mass spectrometer in the analysis of this toxicity biomarker in urine. Due to these characteristics, this approach would constitute an invaluable tool in the clinical laboratory, for measuring TDGA and other toxicity related biomarkers of chemotherapy with proper sensitivity and accuracy.