Acute renal toxicity of sodium chlorate: Redox imbalance, enhanced DNA damage, metabolic alterations and inhibition of brush border membrane enzymes in rats.

Sodium chlorate (NaClO3 ) is widely used in paper and pulp industries and as a non-selective herbicide. Humans can be exposed to NaClO3 through contaminated drinking water due to its improper and unchecked usage in industries and as herbicide. NaClO3 is also present as a major stable by-product in drinking water that has been disinfected with chlorine dioxide. In this study, we have investigated the effect of a single acute oral dose of NaClO3 on rat kidney. Adult male Wistar rats were divided into one control and four NaClO3 treated groups that were orally given different doses of NaClO3 and euthanized 24 hr after the treatment. Oral administration of NaClO3 resulted in increased hydrogen peroxide levels, lipid, and protein oxidation while thiol and glutathione content and activities of brush border membrane enzymes were decreased in kidney in a NaClO3 dose-dependent manner. Significant alterations in the activities of enzymes involved in carbohydrate metabolism and antioxidant defense were also observed. Administration of NaClO3 induced DNA fragmentation and increased DNA-protein cross-linking. Histological studies showed marked damage in kidney from NaClO3 treated animals. These results strongly suggest that NaClO3 induces nephrotoxicity via redox imbalance that results in DNA and membrane damage, metabolic alterations and brush border membrane enzyme dysfunction.

Sodium chlorate, a major water disinfection byproduct, alters brush border membrane enzymes, carbohydrate metabolism and impairs antioxidant system of Wistar rat intestine.

Sodium chlorate (NaClO3 ) is a widely used nonselective herbicide. It is also generated as a by-product during disinfection of drinking water by chlorine dioxide. The purpose of this study was to evaluate the effect of NaClO3 on rat intestine. Adult male rats were randomly divided into five groups: control and remaining four groups were administered orally different doses of NaClO3 and sacrificed 24 h after the treatment. The administration of NaClO3 produced acute oxidative stress in the intestine, which manifested in the form of markedly enhanced malondialdehyde levels and carbonyl content and lowered total sulfhydryl groups and glutathione levels. The activities of several brush border membrane (BBM) enzymes were greatly reduced as compared to control. There were alterations in the activities of various enzymes of carbohydrate metabolism and those involved in maintaining the antioxidant defense system. Histological studies support the biochemical results showing NaClO3 dose-dependent increase in tissue damage. Thus, the present study shows that oral administration of NaClO3 decreases the activities of BBM enzymes, induces oxidative stress, alters metabolic pathways, and impairs the antioxidant system of rat intestine. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1607-1616, 2017.

[How does a pesticide residue turn into a contaminant?] / Wie wird ein Pflanzenschutzmittelrückstand zur Kontaminante?

Over the last few years pesticide residues have been repeatedly detected during official food controls that would not be expected from authorized pesticide uses. These residues do not always pose a health risk for consumers. However, the legal and economic consequences of such findings are often far-reaching, especially if the admissible maximum residue limits have been fixed at the LOQ level only. For some example cases, we discuss the real entry pathways into the food chain and under which circumstances residues of such unintentionally added substances could be better regulated under the contaminant legal framework rather than the pesticide legal framework.

[Effect of conditioned medium from mesenchymal stem cells on regeneration of endothelium at HCl-induced damage trachea in rats].

The purpose. Respiratory epithelium regeneration is studied in rats with tracheal damage induced by inhaling hydrochloric acid vapor. Method. Regeneration process after the chemical burn was activated by intratracheal administration of preparations obtained from the same-species mesenchymal stem cells (MSC). Results. Tracheal epithelium is shown to recover almost completely on day 3-7 after applying MSC compositions (MSCs). Closed structures containing ciliated cells similar to ciliated cells of the respiratory epithelium lining the trachea are formed in the submucosal epithelium during regeneration. These structures migrate towards epithelium and get incorporated into the damaged epithelium. This phenomenon is apparently indicative of the special mechanism of respiratory epithelium regeneration after HCl-induced injury. Conclusion. It is demonstrated in this study that cell-free MSCs instilled intratracheally promote the recovery of normal submucosal epithelium by either preventing or reducing necrosis and inflammation. Such topical MSCs administration significantly accelerates migration of ciliated cell towards the surface and de novo formation of the ciliary epithelium.

Establishment of an in vitro monolayer model of macular corneal dystrophy.

Macular corneal dystrophy (MCD) is characterized by multiple punctate gray-white opacities in the corneal stromal region, due to the accumulation of abnormally sulfated keratan sulfates. We attempted to develop an in vitro model of MCD by simulating the sulfation inhibition using sodium chlorate, a chemical inhibitor of 3'-phosphoadenosine-5'-phosphosulfate (PAPs). The SEM and micro-Raman spectroscopy results showed the hallmark feature of MCD. Further the gene expression studies elucidated the direct effect of sulfation inhibition on the WNT pathway, that in turn downregulated production of matrix metalloproteinases (MMPs), which causes abnormal matrix deposits leading to loss of transparency in vivo. It also resulted in downregulation of integrin and cadherin complexation that leads to disruption of the epithelial layer in the MCD affected corneas. This study offers a promising initial step toward establishing a relevant in vitro MCD disease model, to assess signaling transduction pathways and devise potential treatment strategies based on MMP administration to the MCD affected corneas.

Trihalomethanes, chlorite, chlorate in drinking water and risk of congenital anomalies: a population-based case-control study in Northern Italy.

BACKGROUND: Epidemiological evidence of an association between disinfection by-products (DBPs) exposure via drinking water and reproductive outcomes is still inconclusive. OBJECTIVE: The aim of this study was to investigate the association between trihalomethanes (THMs), chlorite and chlorate exposure and congenital anomalies. METHODS: A case-control study was carried out in Emilia-Romagna Region (Italy). Data on 1917 different congenital anomalies (neural tube, cardiac, diaphragm and abdominal wall, oesophagus, cleft lip and palate, respiratory, urinary tract and chromosomal anomalies) observed in the period 2002-2005 were extracted from the Regional Malformation Registry. Four controls (newborns without anomalies) were randomly selected form the Regional Birth Register and frequency matched to cases according to pregnancy period. The network supplying water during the first trimester of pregnancy was identified on the basis of mother's address: DBPs data, technical and structural information were linked to each subject. RESULTS: Overall, THMs exposure was very low (mean: 3.8±3.6 µg/l), and no risk excess was observed. Chlorite and chlorate values were fairly high (mean: 427±184 µg/l and 283±79 µg/l, respectively). Women exposed to chlorite level >700 µg/l were at higher risk of newborns with renal defects (OR: 3.30; 95% IC: 1.35-8.09), abdominal wall defects (OR: 6.88; 95% IC: 1.67-28.33) and cleft palate (OR: 4.1; 95% IC: 0.98-16.8); women exposed to chlorate level >200 µg/l were at higher risk of newborns with obstructive urinary defects (OR: 2.88; 95% IC: 1.09-7.63), cleft palate (OR: 9.60; 95% IC:1.04-88.9) and spina bifida (OR: 4.94; 95% IC:1.10-22). CONCLUSIONS: This was the first study showing an excess risk of different congenital anomalies related to chlorite and chlorate exposure via drinking water: further research is needed to confirm the observed relationships in large datasets, specifically for chlorate, an unregulated DBP.

Effects of short-term sodium chlorate exposure on pigs.

The present study evaluated the effects of exposure to different doses of sodium chlorate in 10-week-old pigs. Twenty pigs were divided into four equal groups and treated with different doses of sodium chlorate: 0, 125, 250 and 500 mg kg-1 body weight per day via the drinking water for 7 consecutive days. The results showed a significant decrease (P < 0.05) in red blood cell and white blood cell counts, packed cell volume, haemoglobin, blood urea nitrogen (P < 0.001) and creatinine levels, and an increase in aspartate aminotransferase and alanine aminotransferase (P < 0.05) activities in swine administered sodium chlorate at a dose of 500 mg kg-1 body weight per day. The histopathological study revealed increased numbers of vacuoles in the convoluted tubules, tubular necrosis and degeneration of the renal tubular epithelial cells, depletion of nuclei and lobular necrosis of the liver in all pigs treated with sodium chlorate at 500 mg kg-1 body weight per day. Thus, 7-day administration of sodium chlorate at 500 mg kg-1 body weight per day to pigs affects the liver and kidney tissues as well as the haematologic and serum biochemical parameters.

Impact of updated BMD modeling methods on perchlorate and chlorate assessments of human health hazard.

Several exposure limits for perchlorate have been developed based on an early key event, inhibition of radioactive iodide uptake (RAIU) by the thyroid. These assessments have used a variety of definitions of the point of departure. The current assessment revisited the modeling for inhibition of RAIU, using state of the science methods. Bayesian hierarchical modeling was used to account for the repeated measures on the same individuals in the key dataset, and the underlying Beta distribution used for the modeling correctly reflected the bounding of RAIU between 0 and 1. We defined the BMR as a point value of 8% RAIU (rather than a change in RAIU), based on descriptions in the medical literature that RAIU below this value is considered abnormal. Because a definition of the BMR based on the mean response would correspond to about 50% of the population with a response below the BMR at the benchmark dose, we used a hybrid definition of the BMR. That is, the BMD was defined as the dose at which it was estimated that there would be a 10% extra risk in the population of having RAIU of 8% or lower. The resulting point of departure based on the BMDL was 0.03 mg/kg-day.

Expression of the cassava nitrate transporter NRT2.1 enables Arabidopsis low nitrate tolerance.

The cassava grows well on low-nutrient soils because of its high-affinity to absorb nitrate. However, the molecular mechanisms by which cassava adapts itself to this environment remain elusive, although we have cloned a putative gene named MeNRT2.1 which has a crucial role in high-affinity nitrate transporter from cassava seeding. Here, the expression pattern of MeNRT2.1 was further assessed using the GUS activity driven by MeNRT2.1 promoter in Arabidopsis transformation plants. The GUS activity was monitored over time following the reduction of nitrate supply. The GUS gene expression not only peaked in roots after 12 h in 0.2mM nitrate media, but also stained stems and leaves. Arabidopsis plants with overexpression of MeNRT2.1 increased the biomass compared to the wild type on rich nitrogen (N-full) media. However, chlorate sensitivity analysis showed that Arabidopsis plants expressing MeNRT2.1 were more susceptable to chlorate than wild type. Significantly, after growing for 15 days on media containing 0.2mM nitrate concentration, wild-type plants became yellowor died, while the transgenic MeNRT2.1 Arabidopsis plants maintained normal growth. With significant increases in the amount of 15NO- 3 uptake in roots, the MeNRT2.1 plants also increased the contents of chlorophyll and nitrate reductase. Taken together, these results demonstrate that MeNRT2.1 has an important role in adaptation to low nitrate concentration as a nitrate transporter.

Evaluation of chlorite and chlorate genotoxicity using plant bioassays and in vitro DNA damage tests.

In the last few years chlorine dioxide has been increasingly used for disinfecting drinking water in many countries. Although it does not react with humic substances, chlorine dioxide added to water is reduced primarily to chlorite and chlorate ions, compounds that are under investigation for their potential adverse effects on human health. The aim of this research was to study the genotoxicity of chlorite and chlorate and their mixtures. The end-points included two plant tests (chromosomal aberration test in Allium cepa and micronucleus assay in Tradescantia, carried out at different times of exposure) and two genotoxicity tests in human HepG2 cells (comet assay and cytokinesis-blocked micronucleus test). Preliminary toxicity tests were carried out for both plant and HepG2 assays. The results showed that chlorite and chlorate are able to induce chromosomal damage to plant systems, particularly chromosomal aberrations in A. cepa root tip cells, even at concentrations lower than the limit established by Italian normative law and WHO guidelines. In HepG2 cells increased DNA damage was only observed for chlorate at the lowest concentration. No increase in micronuclei frequency was detected in any of the samples tested in human HepG2 cells.

Toxic effects of chlorate on three plant species inoculated with arbuscular mycorrhizal fungi.

Pot experiments were conducted to examine the toxic effects of chlorate on bermudagrass, bahiagrass, and longan seedling with a focus on arbuscular mycorrhizal fungi-plant associations. The results show that application of chlorate could cause slight soil acidification, but the resulting pH was still around 5.5, which is unlikely to adversely affect plant growth. Increase in the application rate of chlorate resulted in a decrease in colonization rate of arbuscular mycorrhizal fungi in plant roots, P uptake by the plants and plant biomass. This appears to suggest that the reduction in plant growth may be related to impeded uptake of P by the plants due to the failure of the plants to form sufficient mycorrhizal associations when chlorate is in sufficient amounts to cause toxicity to arbuscular mycorrhizal fungi. Under the experimental conditions set for this study, bermudagrass suffered from stronger chlorate stress than bahiagrass and longan seedling did in terms of plant-arbuscular mycorrhizal fungi (AMF) symbiosis development.

Sodium chlorate induces DNA damage and DNA-protein cross-linking in rat intestine: A dose dependent study.

Sodium chlorate (NaClO3) is widely used in paper and pulp industries and as a non-selective herbicide. It is also a major by-product generated upon disinfection of drinking water by chlorine dioxide. In this study, we have investigated the genotoxicity of NaClO3 on the small intestine of rats. Adult male rats were divided into 5 groups: one control and four NaClO3 treated groups. The NaClO3 treated groups were given a single acute oral dose of NaClO3 (100, 250, 500 and 750 mg/kg body weight) and sacrificed 24 h later. Administration of NaClO3 caused significant DNA damage in a dose dependent manner in the rat intestine. This was evident from the comet assay which showed DNA strand breaks and was further confirmed by agarose gel electrophoresis and release of free nucleotides. Increased DNA protein cross-linking in NaClO3 administered groups showed formation of a critical lesion which hampers activities of proteins/enzymes involved in DNA repair, transcription and replication. Thus, oral administration of NaClO3 induces DNA damage in the rat intestine, probably through chlorate induced production of reactive oxygen species.

Sodium chlorate, a herbicide and major water disinfectant byproduct, generates reactive oxygen species and induces oxidative damage in human erythrocytes.

Sodium chlorate (NaClO3) is a widely used non-selective herbicide. It is also generated as a byproduct during disinfection of drinking water by chlorine dioxide. In the present work, the effects of NaClO3 on human erythrocytes were studied under in vitro conditions. Incubation of erythrocytes with different concentrations of NaClO3 at 37 °C for 90 min resulted in significant hemolysis. Cell lysates were prepared from NaClO3-treated and untreated (control) erythrocytes and assayed for various biochemical parameters. Methemoglobin levels were significantly increased and methemoglobin reductase activity was reduced upon NaClO3 treatment. There was a significant increase in protein oxidation and lipid peroxidation with a decrease in reduced glutathione and total sulfhydryl content. This suggests the induction of oxidative stress in erythrocytes upon exposure to NaClO3. The occurrence of oxidative stress was confirmed by significantly increased generation of reactive oxygen species and lowered antioxidant response of the cells. NaClO3 treatment also increased nitric oxide levels showing induction of nitrosative stress. The activities of major antioxidant and membrane-bound and metabolic enzymes were significantly altered upon incubation of erythrocytes with NaClO3. The erythrocytes became more osmotically fragile while electron microscopic images showed gross morphological alterations in NaClO3-treated cells. These results show that NaClO3 induces oxidative stress in human erythrocytes, which results in extensive membrane damage and lowers the antioxidant response.

Toxicology and carcinogenesis studies of sodium chlorate (Cas No. 7775-09-9) in F344/N rats and B6C3F1 mice (drinking water studies).

BACKGROUND: Sodium chlorate occurs when drinking water is disinfected by chlorine dioxide. We studied the effects of sodium chlorate in rats and mice to identify potential toxic or carcinogenic hazards to humans. METHODS: We gave groups of male and female rats drinking water containing 125, 1,000, or 2,000 milligrams (mg) of sodium chlorate per liter (L) of water for two years. Male and female mice received 500, 1,000, or 2,000 mg/L. Other groups of animals received plain tap water and served as the control groups. At the end of the study, tissues from more than 40 sites were examined for every animal. RESULTS: Male and female rats receiving sodium chlorate had higher rates of follicular cell hypertrophy of the thyroid gland, and the groups receiving 2,000 mg/L had higher rates of thyroid gland cancer, compared with the control groups. Female mice exposed to sodium chlorate had a few pancreatic islet cell tumors. CONCLUSIONS: We conclude that sodium chlorate caused some thyroid gland neoplasms in male and female rats. The pancreatic islet cell tumors in female mice may have been related to sodium chlorate exposure.

Continuous, low-dose oral exposure to sodium chlorate reduces fecal generic Escherichia coli in sheep feces without inducing clinical chlorate toxicosis.

Our objectives were to determine an effective, yet safe, daily dose of sodium chlorate for reducing fecal shedding of generic Escherichia coli in mature ewes. In a completely randomized experimental design, 25 Targhee ewes (age ∼ 18 mo; BW = 62.5 ± 7.3 kg, mean ± SD) were assigned randomly to 1 of 5 sodium chlorate treatments, which were administered in the drinking water for 5 consecutive days. Treatments were control group (no sodium chlorate) and 4 targeted levels of daily sodium chlorate intake: 30, 60, 90, and 120 mg · kg(-1) BW · d(-1) for 5 d. Individual ewe ad libitum intake of water (with treatments) was measured daily, and BW was measured at the beginning of and 15 and 51 d after the 5-d treatment period. Serum chlorate, whole blood methemoglobin and packed-cell volume (PCV), and fecal generic E. coli and general Enterobacteriaceae coliforms were measured from corresponding samples collected at the end of the 5-d treatment period. Average daily intakes of sodium chlorate from drinking water treatments were 95%, 91%, 90%, and 83% of the target treatment intakes of 30, 60, 90, and 120 mg · kg(-1) BW · d(-1), respectively. Daily sodium chlorate intake remained constant for all treatment groups except for ewes offered 120 mg NaClO3 · kg(-1) BW · d(-1), which decreased (quadratic; P = 0.04) over the course of the 5-d treatment period. This decrease in sodium chlorate intake indicated that the 120-mg NaClO3 level may have induced either toxicity and/or an aversion to the drinking water treatment. Serum chlorate concentrations increased (quadratic; P < 0.001) with increasing sodium chlorate intake. At the end of the 5-d treatment period, mean (least squares ± SEM) serum chlorate concentrations for ewes offered 30, 60, 90, and 120 mg NaClO3 · kg(-1) BW · d(-1) were 15.6 ± 14.1, 32.8 ± 15.8, 52.9 ± 14.1, and 90.3 ± 14.1 µg/mL, respectively. Whole blood methemoglobin and PCV were similar (P = 0.31 to 0.81) among the control group and ewes offered sodium chlorate. Likewise, BW was not affected by sodium chlorate (P > 0.27). Ewes consuming approximately 55 mg NaClO3 · kg(-1) BW · d(-1) or more (i.e., ewes offered 60, 90, and 120 mg) had a >1.4 log unit reduction in fecal E. coli and Enterobacteriaceae coliforms compared with control ewes. We suggest that for a short-term, 5-d dosing strategy, 55 to 81 mg NaClO3 · kg(-1) BW · d(-1) is an effective, yet safe, daily oral dose range for mature ewes to achieve a 97% to 99% reduction in fecal shedding of generic E. coli.

Chlorate poisoning: mechanism of toxicity.

Intoxications with chlorate salts are characterized by methaemoglobin formation, haemolysis and renal insufficiency. The toxic effects on the erythrocyte can be reproduced in vitro. Incubation of human and rabbit erythrocytes with chlorates induces a concentration-dependent oxidation of haemoglobin. This methaemoglobin formation is followed by denaturation of the globin, a cross-linking of erythrocyte membrane proteins and an inactivation of membrane enzymes. The high sensitivity of glucose-6-phosphate dehydrogenase to denaturation by chlorate explains the inefficacy of methylene blue to reduce methaemoglobin formed, as the antidotal effect of methylene blue depends on NADPH formed mainly by the oxidation of glucose-6-phosphate. The observed changes occur only in the presence of methaemoglobin which forms a destabilising complex with chlorate. Methaemoglobin thus autocatalytically increases methaemoglobin formation and destruction of the erythrocyte. As the rabbit is known to have a high methaemoglobin-reduction capacity, human and rabbit erythrocytes were compared. In vitro, the rabbit erythrocyte is less sensitive to oxidative attack than the human red cell. In vivo, an oral dose of sodium chlorate (1 g/kg body wt.) resulted in high serum (16 +/- 4 mM) and urine concentrations (246 +/- 99 mM) in the rabbit. Methaemoglobin was not formed nor could a nephrotoxic effect be observed. These experiments also indicate that the nephrotoxicity of chlorate is mediated by methaemoglobin catalysis.

Erythrocyte membrane alterations as the basis of chlorate toxicity.

The effects of sodium chlorate and of sodium nitrite on human erythrocytes were studied in vitro. Nitrite rapidly oxidised haemoglobin and glutathione; reduction of methaemoglobin (Hbi) by methylene blue was complete during 3 h of incubation with nitrite. With chlorate, a concentration-dependent lag phase was seen before Hbi was formed. After prolonged incubation, Hbi could no longer be reduced with methylene blue. Several other effects were observed that explain the clinical picture of chlorate poisoning which involves haemolysis followed by disseminated intravascular coagulation and renal failure: increased permeability to cations, increased resistance to hypotonic haemolysis and prolonged filtration time through polycarbonate membranes with cylindrical pores of 5 micron diameter. This suggests an increased membrane rigidity due to membrane protein polymerisation, as demonstrated by SDS polyacrylamide gel electrophoresis. Simultaneously, erythrocyte enzymes were inactivated, primarily glucose-6-phosphate dehydrogenase which is necessary for the therapeutic effect of methylene blue. This explains the inefficacy of methylene blue in the treatment of a case of chlorate poisoning that we observed (Arch. Toxicol., 48 (1981) 281).

Effects of the acute rising dose administration of chlorine dioxide, chlorate and chlorite to normal healthy adult male volunteers.

Chlorine dioxide is under consideration for use as a water disinfectant alternative to chlorination in the United States. A rising dose tolerance study was undertaken to assess the relative safety and tolerance of acute administration of chlorine dioxide and its byproducts, chlorite and chlorate, to normal healthy adult male volunteers. In evaluation of an extensive battery of laboratory tests and vital signs, no adverse physiological effects were identified. This provided a data base upon which a controlled 5-month study trial of these substances in normal healthy volunteer subjects was designed.

The effects of chronic administration of chlorine dioxide, chlorite and chlorate to normal healthy adult male volunteers.

The physiological impact of chronic 12 week ingestion of chlorine dioxide and its byproducts, chlorite and chlorate, was compared to the effects of chlorine, chloramine and untreated water. The water disinfectant solutions were administered daily (500 ml, 5 ppm) to normal healthy adult male volunteers. An extensive battery of tests was used to evaluate the physiological impact of the ingested water disinfectants. Upon analysis of both quantitative and qualitative parameters it was concluded that the 12 week chronic administration of chlorine dioxide and its byproducts was accompanied by no clinically important physiological effects.

Strain improvement of chymosin-producing strains of Aspergillus niger var. awamori using parasexual recombination.

Parasexual recombination was used to obtain improved chymosin-producing strains and to perform genetic analysis on existing strains. Chlorate resistance was used to select for a variety of spontaneous nitrate assimilation pathway mutations in strains previously improved for chymosin production using classical strain improvement methods including mutation and screening, and selection for 2-deoxyglucose resistance (dgr). Diploids of these improved strains were generated via parasexual recombination and were isolated on selective media by complementation of nitrate assimilation mutations. A preliminary genetic analysis of diploid and haploid segregants indicated that the dgr trait, resulting in overexpression of chymosin, was recessive. Also, mutations in two different dgr genes resulted in an increased level of chymosin production. When these mutations were combined via parasexual recombination, the resulting haploid segregants produced about 15% more chymosin than either parental strain. CHEF gel electrophoresis was used to determine the chromosomal location of the integrated chymosin DNA sequences, and to verify diploidy in one case where the chromosome composition of two haploid parents differed.