Integrated transcriptomic and metabolomic analysis of the hepatotoxicity of dichloroacetonitrile.

Dichloroacetonitrile (DCAN), an emerged nitrogenous disinfection by-product (N-DBP) in drinking water, has garnered attention owing to its strong cytotoxicity, genotoxicity, and carcinogenicity. However, there are limited studies on its potential hepatotoxicity mechanisms. Understanding hepatotoxicity is essential in order to identify and assess the potential risks posed by environmental pollutants on liver health and to safeguard public health. Here, we investigated the viability, reactive oxygen species (ROS) levels, and cell cycle profile of DCAN-exposed HepG2 cells and analyzed the mechanism of DCAN-induced hepatotoxicity using both transcriptomic and metabolomic techniques. The study revealed that there was a decrease in cell viability, increase in ROS production, and increase in the number of cells in the G2/M phase with an increase in the concentration of DCAN. Omics analyses showed that DCAN exposure increased cellular ROS levels, leading to oxidative damage in hepatocytes, which further induced DNA damage, cell cycle arrest, and cell growth impairment. Thus, DCAN has significant toxic effects on hepatocytes. Integrated analysis of transcriptomics and metabolomics offers new insights into the mechanisms of DCAN-induced hepatoxicity.

Haloacetonitrile stability in cell culture media used in vitro toxicological studies.

Haloacetonitriles (HANs) are an emerging class of nitrogenous disinfection by-products (DBPs) formed in disinfected drinking water and have been reported to be more cyto- and genotoxic than the regulated DBPs. HANs are also known to hydrolyze under neutral pH and normal room temperature. However, the stability of HANs has not been well characterized in DBP toxicological assessments. Most toxicological assessments expose DBPs up to several days which may result in a mixture of HANs and degradation products that might have underestimated HAN toxicity. In this study, HANs stability was characterized in 1) a buffer solution in sealed vials, 2) cell culture media (CCM) in sealed vials, and 3) CCM in 96 sealed well plates with 5% CO2. Solutions were incubated at 37 °C for 3 days. MonoHANs were found to be stable in buffer and CCM except when HANs were incubated in CCM in plates where they could possibly be affected by volatilization and photodegradation during sample handling. However, di- and tri- HANs degraded between 70 and 100% in both buffer solution and CCM. They were also found to be less stable in CCM than in buffer solution possibly from HANs reacting with nucleophiles present in CCM (i.e., amino acids). Identified degradation products include corresponding haloacetamides and haloacetic acids for buffer solutions and only haloacetic acids and an unknown brominated compound for CCM. Results of this study suggests that reported toxicity values might have been underestimated and should consider changing CCM and DBP on a daily basis for a more accurate toxicity measurement.

Protective role of electrophile-reactive glutathione for DNA damage repair inhibitory effect of dibromoacetonitrile.

Dibromoacetonitrile (DBAN) is a disinfection byproduct (DBP) and linked with cancer in rodents, but the mechanism of its carcinogenicity has not been fully elucidated. We recently reported that DBAN induced inhibition of nucleotide excision repair (NER). In this study, we investigated if glutathione (GSH) is involved in the DBAN-induced inhibition of NER. Human keratinocytes HaCaT were pretreated with L-buthionine-(S,R)-sulfoximine (BSO) to deplete intracellular GSH. BSO treatment markedly potentiated the DBAN-induced NER inhibition as well as intracellular oxidation. The recruitment of NER proteins (transcription factor IIH, and xeroderma pigmentosum complementation group G) to DNA damage sites was inhibited by DBAN, which was further exacerbated by BSO treatment. Our results suggest that intracellular GSH protects cells from DBAN-induced genotoxicity including inhibition of DNA damage repair.

Cytogenotoxic evaluation of the acetonitrile extract, citrinin and dicitrinin-A from Penicillium citrinum.

Endophytic fungi are promising sources of bioactive substances; however, their secondary metabolites are toxic to plants, animals, and humans. This study aimed toevaluate the toxic, cytotoxic, mutagenic and oxidant/antioxidant activities of acetonitrile extract (AEPc), citrinin (CIT) and dicitrinin-A (DIC-A) of Penicillium citrinum. For this, the test substances at 0.5; 1.0; 1.5 and 2 µg/mLwere exposed for 24 and 48 h in Artemia salina, and 48 h in Allium cepa test systems. The oxidant/antioxidant test was evaluated in pre-, co- and post-treatment with the stressor hydrogen peroxide (H2O2) in Saccharomyces cerevisiae. The results suggest that the AEPc, CIT and DIC-A at 0.5; 1.0; 1.5 and 2 µg/mL showed toxicity in A. saline, with LC50 (24 h) of 2.03 µg/mL, 1.71 µg/mL and 2.29 µg/mL, and LC50 (48 h) of 0.51 µg/mL, 0.54 µg/mL and 0.54 µg/mL, respectively.In A. cepa, the test substances also exerted cytotoxic and mutagenic effects. The AEPc, CIT and DIC-A at lower concentrations modulated the damage induced by H2O2 in the proficient and mutant strains of S. cerevisiae for cytoplasmic and mitochondrial superoxide dismutase. Moreover, the AEPc at 2 µg/mL and CIT at the two highest concentrations did not affect the H2O2-induced DNA damage in the test strains. In conclusion, AEPc, CIT and DIC-A of P. citrinum may exert their toxic, cytotoxic and mutagenic effects in the test systems possibly through oxidative stress induction pathway.

Comparative Quantitative Toxicology and QSAR Modeling of the Haloacetonitriles: Forcing Agents of Water Disinfection Byproduct Toxicity.

The haloacetonitriles (HANs) is an emerging class of nitrogenous-disinfection byproducts (N-DBPs) present in disinfected drinking, recycled, processed wastewaters, and reuse waters. HANs were identified as primary forcing agents that accounted for DBP-associated toxicity. We evaluated the toxic characteristics of iodoacetonitrile (IAN), bromoacetonitrile (BAN), dibromoacetonitrile (DBAN), bromochloroacetonitrile (BCAN), tribromoacetonitrile (TBAN), chloroacetonitrile (CAN), dichloroacetonitrile (DCAN), trichloroacetonitrile (TCAN), bromodichloroacetonitrile (BDCAN), and chlorodibromoacetonitrile (CDBAN). This research generated the first quantitative, comparative analyses on the mammalian cell cytotoxicity, genotoxicity and thiol reactivity of these HANs. The descending rank order for HAN cytotoxicity was TBAN ≈ DBAN > BAN ≈ IAN > BCAN ≈ CDBAN > BDCAN > DCAN ≈ CAN ≈ TCAN. The rank order for genotoxicity was IAN ≈ TBAN ≈ DBAN > BAN > CDBAN ≈ BDCAN ≈ BCAN ≈ CAN ≈ TCAN ≈ DCAN. The rank order for thiol reactivity was TBAN > BDCAN ≈ CDBAN > DBAN > BCAN > BAN ≈ IAN > TCAN. These toxicity metrics were associated with membrane permeability and chemical reactivity. Based on their physiochemical parameters and toxicity metrics, we developed optimized, robust quantitative structure activity relationship (QSAR) models for cytotoxicity and for genotoxicity. These models can predict cytotoxicity and genotoxicity of novel HANs prior to analytical biological evaluation.

Low-concentration of dichloroacetonitrile (DCAN) in drinking water perturbs the health-associated gut microbiome and metabolic profile in rats.

Dichloroacetonitrile (DCAN) is one of the emerging nitrogenous disinfection by-products (DBPs) in drinking water. However, its potential toxicological effects remain poorly understood, especially at a low concentration found in the environment. In the present study, we investigated whether the consumption of low-concentration DCAN through drinking water would produce significant effects in male SD rats, with particular focus on their physiological traits and changes in their gut microbiome and metabolite profiles. After a 4-weeks DCAN intervention, significant changes were observed in the body weight, blood indices, and histology in DCAN-treated (100 µg/L) group. Proteobacteria was relatively less abundant in 20 and 100 µg/L DCAN-treated groups compared with that in the control group at phylum level. At genus level, Parasutterella and Anaerotruncus were significantly less abundant in both 20 and 100 µg/L DCAN-treated groups than that in the control group. Furthermore, the gut microbiota-related metabolites were dramatically perturbed after DCAN consumption. In the 20 and 100 µg/L DCAN-treated groups, there were 48 and 95 altered metabolites, respectively, and were found to be involved in sphingolipid signaling pathway, fatty acid biosynthesis, and cGMP-PKG signaling pathway. In summary, we demonstrated that consumption of low-concentration DCAN through drinking water could impair host health and induce gut microbiota dysbiosis and gut microflora-related metabolic disorders in male SD rats. Our findings highlight the potential toxicity of low-concentration DBPs and provide new insight into potential causal relationship between low concentration DBPs found in the drinking water and the host health.

Transmission blocking sugar baits for the control of Leishmania development inside sand flies using environmentally friendly beta-glycosides and their aglycones.

BACKGROUND: The sand fly Lutzomyia longipalpis is the main vector of American visceral leishmaniasis, a disease caused by parasites of the genus Leishmania. Adults of this insect feed on blood (females only) or sugar from plant sources, but their digestion of carbohydrates is poorly studied. Beta-glycosides as esculin and amygdalin are plant compounds and release toxic compounds as esculetin and mandelonitrile when hydrolyzed. Beta-glucosidase and trehalase are essential enzymes in sand fly metabolism and participate in sugar digestion. It is therefore possible that the toxic portions of these glycosides, released during digestion, affect sand fly physiology and the development of Leishmania. RESULTS: We tested the oral administration to sand flies of amygdalin, esculin, mandelonitrile, and esculetin in the sugar meal. These compounds significantly decreased the longevity of Lutzomyia longipalpis females and males. Lutzomyia longipalpis adults have significant hydrolytic activities against esculin and feeding on this compound cause changes in trehalase and ß-glucosidase activities. Female trehalase activity is inhibited in vitro by esculin. Esculin is naturally fluorescent, so its ingestion may be detected and quantified in whole insects or tissue samples stored in methanol. Mandelonitrile neither affected the amount of sugar ingested by sand flies nor showed repellent activity. Our results show that mandelonitrile significantly reduces the viability of L. amazonensis, L. braziliensis, L. infantum and L. mexicana, in a concentration-dependent manner. Esculetin caused a similar effect, reducing the number of L. infantum and L. mexicana. Female L. longipalpis fed on mandelonitrile had a reduction in the number of parasites and prevalence of infection after seven days of infection with L. mexicana, either by counting in a Neubauer chamber or by qPCR assays. CONCLUSIONS: Glycosides have significant effects on L. longipalpis longevity and metabolism and also affect the development of parasites in culture and inside the insect. These observations might help to conceptualize new vector control strategies using transmission blocking sugar baits.

Single and combined effects of selected haloacetonitriles in a human-derived hepatoma line.

Haloacetonitriles (HANs) are nitrogenous disinfection byproducts (N-DBPs) detected in drinking water that have high toxicity and are a high risk to human health. The cytotoxicity and genotoxicity as well as the oxidative stress of five HANs, namely chloroacetonitrile (CAN), dichloroacetonitrile (DCAN), trichloroacetonitrile (TCAN), bromoacetonitrile (BAN), and dibromoacetonitrile (DBAN) on a hepatoma cell line (HepG2) were determined by single, binary or ternary exposure. The median effective concentrations, based on cell viability, ranged from 0.8360 mg/L for BAN to 256.9 mg/L for DCAN, with a cytotoxicity order of BAN > DBAN > CAN > TCAN > DCAN. The lowest observed effective concentrations regarding DNA damage were 0.01 mg/L for CAN and DCAN, 0.1 mg/L for DBAN and TCAN, and 1 mg/L for BAN. The DNA damage induced by CAN, DCAN and TCAN was repaired to about 80% in 30 min, and when induced by BAN and DBAN, it was repaired completely in 60 min. The intracellular reactive oxygen species (ROS) levels were significantly increased by the five HANs, and bromo-acetonitrile produced a stronger oxidative stress than chloro-acetonitrile. Co-exposure of DCAN, TCAN and DBAN significantly inhibited cell viability, induced DNA damage and facilitated ROS generation in HepG2 cells. However, the interactive effects were inconsistent for the different endpoints, which seemed to be antagonism for cell viability but synergy for ROS generation.

Cellular adverse actions of dibromoacetonitrile, a by-product in water bacterial control, at sublethal levels in rat thymocytes.

The aim of this study was to investigate the effects of dibromoacetonitrile (DBAN), a by-product in water bacterial control, at sublethal concentrations on rat thymocytes, by using a cytometric technique with appropriate fluorescent dyes. By using this method, the possibility that DBAN induces cellular actions related to oxidative stress was assessed. DBAN reduced the content of cellular nonprotein thiols under Zn2+-free conditions. It elevated the intracellular level of Zn2+, being independent from external Zn2+. DBAN increased cell vulnerability to the cytotoxic action of hydrogen peroxide. These actions of DBAN were likely related to oxidative stress. DBAN is formed by the reaction of bromides and chlorinated oxidants during water disinfection. Hydrolysis of 2,2-dibromo-3-nitrilopropionamide, an antimicrobial used in hydraulic fracturing fluids for production of shale gas and oil, produces DBAN. Therefore, the concern regarding the levels of DBAN in industrial water systems is necessary to avoid the environmental risk to humans and wild mammals.

Formation and estimated toxicity of trihalomethanes, haloacetonitriles, and haloacetamides from the chlor(am)ination of acetaminophen.

The occurrence of pharmaceuticals and personal care products (PPCPs) in natural waters, which act as drinking water sources, raises concerns. Moreover, those compounds incompletely removed by treatment have the chance to form toxic disinfection byproducts (DBPs) during subsequent disinfection. In this study, acetaminophen (Apap), commonly used to treat pain and fever, was selected as a model PPCP. The formation of carbonaceous and nitrogenous DBPs, namely trihalomethanes, haloacetonitriles, and haloacetamides, during chlor(am)ination of Apap was investigated. Yields of chloroform (CF), dichloroacetonitrile (DCAN), dicholoacetamide (DCAcAm), and tricholoacetamide (TCAcAm), during chlorination were all higher than from chloramination. The yields of CF continuously increased over 48h during both chlorination and chloramination. During chlorination, as the chlorine/Apap molar ratios increased from 1 to 20, CF yields increased from 0.33±0.02% to 2.52±0.15%, while the yields of DCAN, DCAcAm and TCAcAm all increased then decreased. In contrast, during chloramination, increased chloramine doses enhanced the formation of all DBPs. Acidic conditions favored nitrogenous DBP formation, regardless of chlorination or chloramination, whereas alkaline conditions enhanced CF formation. Two proposed formation mechanisms are presented. The analysed DBPs formed during chlorination were 2 orders of magnitude more genotoxic and cytotoxicity than those from chloramination.

Evaluation of the water disinfection by-product dichloroacetonitrile-induced biochemical, oxidative, histopathological, and mitochondrial functional alterations: Subacute oral toxicity in rats.

Dichloroacetonitrile (DCAN), an emerging nitrogenous disinfection by-product, is more genotoxic and cytotoxic than the currently regulated carbonaceous disinfection by-products such as haloacetic acids. Few mechanistic studies have been conducted on the hepatic and renal toxicities of DCAN. This study examined the clinical biochemical, hematological, histopathological, oxidative, and mitochondrial functional alterations to evaluate the systematic toxicity after subacute oral exposure of 11 or 44 mg/kg/day in rats for 28 days. Body and spleen weights were lower, and organ-to-body weight ratios of the liver and kidney were higher in rats administered 44-mg/kg DCAN than in controls. The activities of serum alanine aminotransferase and alkaline phosphatase, and concentrations of blood serum urea nitrogen and retinol-binding protein were increased in rats administered 44-mg/kg DCAN compared with those of controls, thereby indicating hepatic and renal damage in this group. This was confirmed by histopathological alterations, including hepatic sinus dilation, extensive hemorrhage, vacuolar degeneration in the liver and glomerulus hemorrhage, and renal tubular swelling, in DCAN-exposed rats. Exposure to 44-mg/kg DCAN induced hepatic oxidative damage shown by the significant increase in malonaldehyde levels, a poisonous product of lipid peroxidation. Exposure to 44-mg/kg DCAN significantly increased hepatic glutathione content and mitochondrial bioenergy as noted by the elevation of mitochondrial membrane potential and cytochrome c oxidase activity, which might be attributed to compensatory pathophysiologic responses to DCAN-induced hepatic mitochondrial damage.

The drinking water contaminant dibromoacetonitrile delays G1-S transition and suppresses Chk1 activation at broken replication forks.

Chlorination of drinking water protects humans from water-born pathogens, but it also produces low concentrations of dibromoacetonitrile (DBAN), a common disinfectant by-product found in many water supply systems. DBAN is not mutagenic but causes DNA breaks and elevates sister chromatid exchange in mammalian cells. The WHO issued guidelines for DBAN after it was linked with cancer of the liver and stomach in rodents. How this haloacetonitrile promotes malignant cell transformation is unknown. Using fission yeast as a model, we report here that DBAN delays G1-S transition. DBAN does not hinder ongoing DNA replication, but specifically blocks the serine 345 phosphorylation of the DNA damage checkpoint kinase Chk1 by Rad3 (ATR) at broken replication forks. DBAN is particularly damaging for cells with defects in the lagging-strand DNA polymerase delta. This sensitivity can be explained by the dependency of pol delta mutants on Chk1 activation for survival. We conclude that DBAN targets a process or protein that acts at the start of S phase and is required for Chk1 phosphorylation. Taken together, DBAN may precipitate cancer by perturbing S phase and by blocking the Chk1-dependent response to replication fork damage.

Tolerance to dichloroacetonitrile-induced neurotoxicity in streptozotocin-induced diabetic rats.

Diabetes mellitus has potential to alter the toxicity of hazardous chemicals. Dichloroacetonitrile (DCAN) is one of high-risk nitrogenous disinfection by-products. This study evaluated the neurotoxicity of DCAN (11, 44 and 88mg/kg) in normoglycaemic and streptozotocin (STZ)-induced diabetic rats via orally for 28days. STZ diabetes prolonged the median survival time and total lethal time after DCAN (88mg/kg) exposure when compared with that observed in normoglycaemic rats. DCAN altered motor activity and induced anxiety behaviour in normoglycaemic rats; but it did not exaggerate behavioural changes in STZ diabetic rats. DCAN -induced brain oxidative damage by compensatory increase glutathione content and decrease malonaldehyde levels; but it did not induce oxidative damage in diabetic rats. STZ diabetes slowed down the pathological pace of DCAN-induced brain mitochondrial dysfunction by decreasing reactive oxygen species and increasing cytochrome C oxidase activity. In conclusion, the present study indicated that STZ diabetic rats are resistant to DCAN-induced neurotoxicity at the dosage and with the dosage schedule in 28-day subacute toxicity test.

Investigation of nuclear enzyme topoisomerase as a putative molecular target of monohaloacetonitrile disinfection by-products.

Disinfection by-products occur widely as the unintended effect of water disinfection and are associated with toxicity and adverse human health effects. Yet the molecular mechanisms of their toxicity are not well understood. To investigate the molecular basis of hyperploidy induction by monohaloacetonitriles, the interaction of monohaloacetonitriles with topoisomerase II in Chinese hamster ovary cells was examined. We showed a concentration-dependent inhibition of DNA decatenation activity of topoisomerase under acellular conditions while in vitro monohaloacetonitrile treatment expressed mixed results. The working hypothesis, that topoisomerase II is a molecular target of monohaloacetonitriles, was only partially supported. Nevertheless, this research serves as a starting point toward molecular mechanisms of toxic action of monohaloacetonitriles.

Dichloroacetonitrile induces cytotoxicity through oxidative stress-mediated and p53-dependent apoptosis pathway in LO2 cells.

Dichloroacetonitrile (DCAN), one of the disinfection byproducts of water chlorination, induces cell proliferation and apoptosis; however, the detailed mechanism remains unclear. Oxidative stress participates in various biological processes, including DNA damage and cytotoxicity. To explore whether oxidative stress mediated DCAN-induced cell proliferation and apoptosis, we assessed the effect of redox imbalance and apoptosis in LO2 cells. We observed increase of reactive oxygen species and malondialdehyde and increased apoptosis by 13.6% in 500 µM DCAN compared with the control group. We also observed a decrease of antioxidant ability damage including glutathione, superoxide dismutase, and total antioxidant capacity depletion. Furthermore, DCAN might activate oxidative stress-mediated apoptosis pathway via up-regulation of p53 expression and caspase-3 activity. Therefore, we conclude that DCAN may activate apoptotic signals via p53 up-regulation and oxidative stress-mediated apoptosis in LO2 cells.

Metabolomics evaluation of the in vivo toxicity of bromoacetonitriles: One class of high-risk nitrogenous disinfection byproducts.

Bromoacetonitriles (BANs), one class of nitrogenous disinfection byproducts (N-DBPs), have frequently been detected in drinking water. The cytotoxicity and genotoxicity of BANs have been demonstrated in mammalian cells. However, a systematic study of the in vivo toxicity of BANs is rare. In this study, metabolomics combined with histopathology and oxidative stress analysis were used to evaluate the toxicity of BANs in mice. The results indicated that BAN exposure induced liver and kidney injury in mice. Furthermore, the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities decreased, and the level of malonaldehyde (MDA) increased in mice livers due to BANs exposure, which indicated that hepatic oxidative stress was induced. These toxicities increased with an increasing number of bromine at the α carbon. In addition, BAN exposure disrupted the metabolic pathways of amino acid, energy and lipid metabolism in mice. Our results provide evidence for the comprehensive omics endpoints of the in vivo toxicity of BANs.

Detection of genotoxic effects of drinking water disinfection by-products using Vicia faba bioassay.

Plant-based bioassays have gained wide use among the toxicological and/or ecotoxicological assessment procedures because of their simplicity, sensitivity, low cost, and reliability. The present study describes the use of Vicia faba (V. faba) micronucleus (MN) test and V. faba comet assay in the evaluation of the genotoxic potential of disinfection by-products (DBPs) commonly found in chlorine-disinfected drinking water. Five haloacetic acids and three halogenated acetonitriles were chosen as representatives of DBPs in this study because they are of potentially great public health risk. Results of the MN test indicated that monochloroacetic acid (MCA), monobromoacetic acid (MBA), dichloroacetic acid (DCA), dibromoacetic acid (DBA), trichloroacetic acid (TCA), and trichloroacetonitrile (TCAN) caused a statistically significant increase in MN frequency in V. faba root tip cells. However, no genotoxic response was observed for dichloroacetonitrile (DCAN) and dibromoacetonitrile (DBAN). Results of the comet assay showed that all tested DBPs induced a statistically significant increase in genomic DNA damage to V. faba root tip cells. On considering the capacity to detect genomic damage of a different nature, we suggest that a combination of V. faba MN test and V. faba comet assay is a useful tool for the detection of genotoxic effects of DBPs. It is worthy of assessing the feasibility of using V. faba comet assay combined with V. faba MN test to screen for the genotoxic activity of chlorinated drinking water in future work.

Acute toxicity of dichloroacetonitrile (DCAN), a typical nitrogenous disinfection by-product (N-DBP), on zebrafish (Danio rerio).

Dichloroacetonitrile (DCAN) is a typical nitrogenous disinfection by-product (N-DBP) and its toxicity on aquatic animals is investigated for the first time. The present study was designed to investigate the potential adverse effects of DCAN on zebrafish. DCAN could induce developmental toxicity to zebrafish embryos. A significant decrease in hatchability and an increase in malformation and mortality occurred when DCAN concentration was above 100µg/L. Heart function alteration and neuronal function disturbance occurred at concentration higher than 500 and 100µg/L, respectively. Further, DCAN was easily accumulated in adult zebrafish. The rank order of declining bioconcentration factor (BCF) was liver (1240-1670)> gill (1210-1430)> muscle (644-877). DCAN caused acute metabolism damage to adult zebrafish especially at 8 days exposure, at which time the "Integrated Biomarker Response" (IBR) index value reached 798 at 1mg/L DCAN dose. Acute DNA damage was induced to adult zebrafish by DCAN even at 10µg/L dose.