Spectral shift supported epichlorohydrin toxicity and the protective role of sage.

In this study, the toxicity of epichlorohydrin, a chemical intermediate, was investigated by using Allium cepa L. test material as a bio-indicator. In addition, the protective role of sage leaf extract (Slex) against this toxicity was investigated. Toxicity was handled with the help of physiological (germination percentage, root elongation, and weight gain), cytogenetic (mitotic index = MI, micronucleus = MN, and chromosomal abnormalities = CAs), biochemical (malondialdehyde = MDA, superoxide dismutase = SOD, and catalase = CAT), and anatomical (root meristem cell damages) parameters. A. cepa bulbs were divided into 6 groups (1 control, 5 applications). The bulbs in the control group were treated with tap water, and the bulbs in the application group were treated with epichlorohydrin at a dose of 100 mg/L and Slex at two different doses (190 mg/L and 380 mg/L) and germinated. Germination process was continued uninterruptedly for 72 h in all groups. At the end of the period, physiological parameter measurements were carried out in the bulbs. In addition, root tips were collected and made ready for cytogenetic, biochemical, and anatomical measurements and microscopic observations. As a result, exposure to epichlorohydrin caused statistically significant (p < 0.05) decreases in germination percentage, root length, weight gain, and MI, and statistically significant (p<0.05) increases in MN frequency, CA numbers, MDA level, SOD, and CAT enzyme activities. Epichlorohydrin exposure induced CAs such as fragment, sticky chromosome, unequal distribution of chromatin, reverse polarization, and disordered mitosis in root meristem cells. The toxicity of epichlorohydrin was due to its interaction with DNA, and this interaction was confirmed by the spectral shift in the DNA spectrum. In addition, epichlorohydrin caused anatomical damages such as epidermis cell damage, cortex cell damage, thickening of the cortex cell wall, and flattened cell nuclei in root meristem cells. The application of Slex together with epichlorohydrin decreased the toxicity of epichlorohydrin and again caused statistically significant (p < 0.05) improvements in the values of all the parameters examined. In other words, germination percentage, root length, weight gain, and MI increased again and MN frequency, CAs numbers, MDA level, SOD, and CAT enzyme activities decreased. It was determined that this improvement was even more pronounced at 380 mg/L dose of Slex. As a result, it was determined that epichlorohydrin caused multiple-toxicity for the investigated indicator organism, and Slex had a reducing role in this toxicity. For this reason, Slex should be included in the daily diet as an antioxidant beverage in order to protect from the toxicity of chemical agents exposed in daily life or to reduce their effects.

Cross-linking by epichlorohydrin and diepoxybutane correlates with cytotoxicity and leads to apoptosis in human leukemia (HL-60) cells.

The bifunctional alkylating agents epichlorohydrin (ECH) and diepoxybutane (DEB) have been linked to increased cancer risks in industrial workers. These compounds react with DNA and proteins, leading to genotoxic effects. We used the comet assay to monitor formation of cross-links in HL-60 cells treated with ECH, DEB, and the structurally related anti-cancer drug mechlorethamine (HN2). We report a time- and dose-dependent cytotoxicity that correlated with cross-linking activity, following the order HN2 > DEB > ECH. The rate of cross-link repair also varied with drug, with ECH-induced lesions the fastest to repair. High drug doses led to the formation of saturating amounts of HN2 cross-links that were repaired inefficiently. DEB and ECH produced fewer overall cross-links, but some were also resistant to repair. These persistent cross-links may activate cell-cycle arrest to allow repair of damage, with prolonged arrest triggering apoptosis. Quantitative reverse transcription polymerase chain reaction experiments revealed that treatment of HL-60 cells with DEB and ECH results in up-regulation of several genes involved in the intrinsic (mitochondrial) apoptosis pathway, including BAX, BAK1, CASP-9, APAF-1, and BCL-2. These findings contribute to our understanding of the principles underlying the carcinogenic potentials of these xenobiotics.

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.

What are the manifestations of ocular exposure to epichlorohydrin?

The Occupational Medicine Forum is prepared by the ACOEM Occupational and Environmental Medical Practice Committee and does not necessarily represent an official ACOEM position. The Forum is intended for health professionals and is not intended to provide medical or legal advice, including illness prevention, diagnosis or treatment, or regulatory compliance. Such advice should be obtained directly from a physician and/or attorney.

Lethal and sublethal toxicity of the industrial chemical epichlorohydrin on Rhinella arenarum (Anura, Bufonidae) embryos and larvae.

Lethal and sublethal toxicity of the major chemical used in epoxide compounds, epichlorohydrin (ECH) was evaluated on the early life cycle of the common South American toad, Rhinella arenarum (Anura, Bufonidae). The stages evaluated were (according to Del Conte and Sirlin): early blastula (S.3-S.4), gastrula (S.10-S.12), rotation (S.15), tail bud (S.17), muscular response (S.18), gill circulation (S.20), open mouth (S.21), opercular folds (S.23) and complete operculum (S.25). The LC50 and EC50 values for lethal and sublethal effects were calculated. The early blastula was the most sensitive stage to ECH both for continuously and pulse-exposures (LC50-24h=50.9 mg L(-1)), while S.20 was the most resistant (LC50-24h=104.9 mg L(-1)). Among sublethal effects, early blastula was also the most sensitive stage (LOEC-48 h=20 mg L(-1)) and it has a Teratogenic Index of 2.5, which indicates the teratogenic potential of the substance. The main abnormalities were persistent yolk plugs, cell dissociation, tumors, hydropsy, oral malformations, axial/tail flexures, delayed development and reduced body size. ECH also caused neurotoxicity including scarce response to stimuli, reduction in the food intake, general weakness, spasms and shortening, erratic or circular swimming. Industrial contamination is considered an important factor on the decline of amphibian populations. Considering the available information about ECH's toxicity and its potential hazard to the environment, this work shows the first results of its developmental toxicity on a native amphibian species, Rhinella arenarum.

Apoptotic cell death in rat epididymis following epichlorohydrin treatment.

Epichlorohydrin (ECH) is an antifertility agent that acts both as an epididymal toxicant and an agent capable of directly affecting sperm motility. This study identified the time course of apoptotic cell death in rat epididymides after ECH treatment. Rats were administrated with a single oral dose of ECH (50 mg/kg). ECH-induced apoptotic changes were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and its related mechanism was confirmed by Western blot analysis and colorimetric assay. The TUNEL assay showed that the number of apoptotic cells increased at 8 h, reached a maximum level at 12 h, and then decreased progressively. The Western blot analysis demonstrated no significant changes in proapoptotic Bcl-2-associated X (Bax) and anti-apoptotic Bcl-2 expression during the time course of the study. However, phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) and phospho-c-Jun amino-terminal kinase (p-JNK) expression increased at 8-24 h. Caspase-3 and caspase-8 activities also increased at 8-48 h and 12-48 h, respectively, in the same manner as p-p38 MAPK and p-JNK expression. These results indicate that ECH induced apoptotic changes in rat epididymides and that the apoptotic cell death may be related more to the MAPK pathway than to the mitochondrial pathway.

One-generation reproductive toxicity study of epichlorohydrin in Sprague-Dawley rats.

This study was conducted to evaluate the potential reproductive toxicity of epichlorohydrin in a one-generation reproduction toxicity study in compliance with OECD Test Guideline 415. Twenty-four male and female rats per group were given epichlorohydrin by gavage at 0, 3.3, 10, and 30 mg/kg/day. Males were dosed for 10 weeks prior to and during mating. Females were dosed from 2 weeks before mating to day 21 of lactation. At 30 mg/kg, an increase in the incidence of clinical signs (i.e., nasal discharge, soft feces, depression, and piloerection), gross necropsy findings (i.e., cystic pustule of the epididymidis and enlargement of the kidney) and the weights of heart, liver, and epididymidis, a decrease in male fertility, and an increased incidence of histopathological changes of the testis, epididymidis, and kidney were observed. At 10 mg/kg, decreased male fertility and increased kidney weight and incidence of histopathological changes of the epididymidis were found. There was a slight, but nonsignificant, reduction in the male fertility index at the dose of 3.3 mg/ kg. Under these experimental conditions, the lowest-observed-adverse-effect level of epichlorohydrin was 3.3 mg/kg/day for parent animals and their offspring. The absolute toxic dose for parent animals and their offspring was estimated to be 10 mg/kg/day.

Induction of oxidative stress in the epididymis of rats after subchronic exposure to epichlorohydrin.

This study investigated the effects of epichlorohydrin (ECH) on spermatogenesis and antioxidant system in rats. An increase in the incidence of clinical signs, gross pathology and histopathology findings in the epididymidis, and sperm abnormalities and a decrease in the testicular spermatid counts, epididymal sperm counts, and sperm motility were observed at 30 mg/kg/day. Oxidative stress in the epididymal tissue was detected at > or =3.3 mg/kg/day. The results show that graded doses of ECH elicit depletion of antioxidant defense system and that the adverse effects on male reproductive function in ECH-treated rats may be due to the induction of oxidative stress.

Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA.

The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.

Effect of O6-alkylguanine-DNA alkyltransferase on genotoxicity of epihalohydrins.

The effect of O(6)-alkylguanine-DNA alkyltransferase (AGT) on the toxicity and mutagenicity of epihalohydrins was studied. AGT is a DNA repair protein that protects cells from agents that produce genotoxic O(6)-alkylguanine lesions by transferring the alkyl group to an internal cysteine residue (Cys(145) in human AGT) in a single-step. This cysteine acceptor site is highly reactive and epihalohydrins reacted readily with AGT at this site with a halide order of reactivity of Br > Cl > F. AGT expression in bacterial cells caused a very large increase in the mutagenicity and cytotoxicity of epibromohydrin. The mutations were almost all G:C to A:T transitions. Epichlorohydrin also augmented AGT-mediated mutagenesis but to a lesser extent than epibromohydrin. In vitro experiments showed that AGT was covalently cross-linked to DNA in the presence of epibromohydrin and that this conjugation occurred predominantly at Cys(145), and to a smaller extent at Cys(150), a less reactive residue also located within the active site pocket. Two pathways yielding the AGT-DNA adduct were found to occur. The predominant mechanism results in an AGT-epihalohydrin intermediate, which, facilitated by the DNA binding properties of AGT, then reacts covalently with DNA. The second pathway involves an initial reactive DNA-epihalohydrin intermediate that subsequently reacts with AGT. Our results show that the paradoxical AGT-mediated increase in genotoxicity which has previously been shown to occur with dihaloalkanes, butadiene diepoxide and nitrogen mustards, also occurs with epihalohydrins and is likely to contribute to their toxicity and mutagenicity.

Quantification of N-(3-chloro-2-hydroxypropyl)valine in human haemoglobin as a biomarker of epichlorohydrin exposure by gas chromatography-tandem mass spectrometry with stable-isotope dilution.

Epichlorohydrin (ECH) is an important industrial intermediate for the production of polymers and surface coatings. Animal experiments support the classification of ECH as a carcinogen, and a significant contribution to the cancer risk of ECH exposed humans has to be considered. Upon uptake, epichlorohydrin reacts with nucleophilic moieties of N- and S-containing macromolecules to form stable adducts, e.g. with haemoglobin. In this article, we describe a GC-tandem MS method for the quantitative analysis of the primary ECH adduct to the N-terminal amino acid of human haemoglobin, i.e. of N-(3-chloro-2-hydroxypropyl)valine (CHPV), using a globin labelled with d(5)-ECH as the internal standard. Incubation of erythrocyte lysate from human blood with ECH or d(5)-ECH yielded two reaction products, with CHPV being the major component. The GC-tandem MS method is based on a modified Edman degradation procedure with subsequent O-acetylation. The limits of detection and quantification of this method are 10 and 25 pmol/g globin, respectively. Intra- and inter-assay imprecision of the method was about 12 and 15%, respectively, and the mean recovery was 105 and 96% at the levels of 25 and 100 pmol of CHPV per g globin, respectively. The present study reports for the first time on the analysis of CHPV as a haemoglobin adduct of ECH using GC-tandem MS and a stable-isotope labelled internal standard. By this method we quantified haemoglobin adducts of ECH in the blood of subjects potentially exposed to ECH after a freight train accident. Our study points to CHPV in human haemoglobin as a possible biomarker for epichlorohydrin exposure.

DNA interstrand cross-linking by epichlorohydrin.

Epichlorohydrin (ECH), an important industrial chemical, is a bifunctional alkylating agent with the potential to form DNA cross-links. Occupational exposure to this suspect carcinogen leads to chromosomal aberrations, and ECH has been shown previously to undergo reaction with DNA in vivo and in vitro. We used denaturing polyacrylamide gel electrophoresis to monitor the possible formation of interstrand cross-links within DNA oligomers by ECH and the related compound, epibromohydrin (EBH). Although both compounds did indeed form cross-links between deoxyguanosine residues, EBH was a more efficient cross-linker than ECH. The optimal pH for cross-linking also varied, with ECH more efficient at pH 5.0 and EBH more efficient at pH 7.0. Both agents were relatively flexible in the sequences targeted, with comparable efficiencies for 5'-GGC and 5'GC sites. Furthermore, interstrand cross-linking by the two optical isomers of ECH correlated with their relative cytotoxicities, with R-ECH about twice as potent as S-ECH.

In vivo assessment of epichlorohydrin effects: the chorioallantoic membrane model.

BACKGROUND: Previous studies on the effects of the epichlorohydrin (ECH) epoxide demonstrated this compound's toxicity and mutagenicity and suggested a carcinogenic activity also in humans. To gain a better understanding of ECH effects in vivo, the substance was tested on developing tissues utilizing the chick embryo chorioallantoic membrane (CAM) assay. MATERIAL/METHODS: Gelatin sponges adsorbed with ECH were implanted onto nine-day CAMs. After five days the membranes were fixed, cut in serial sections, and stained with toluidine blue. Sections of the ECH-treated CAMs were also submitted to immunocytochemistry for the basal lamina glycoprotein laminin and the gap junction protein connexin 43 (Cx43). Control CAMs were treated with saline solution and submitted to identical procedures. RESULTS: ECH-treated CAMs displayed proliferation of both the epithelial layers and the mesenchyme cells and vessels. The laminin immunolabeling was interrupted beneath the ectoderm thickenings, which penetrated the mesenchyme. The endoderm showed papilloma-like formations and its laminin-positive basal membrane protruded toward the mesenchyme, together with clusters of endodermal cells. The mesenchyme showed increased numbers of cells and microvessels. These reactions were restricted to regions corresponding to the implant. Cx43 expression was strongly decreased in the ECH-treated CAMs compared with the controls, where the connexin punctate pattern regularly decorated the epithelial cell contours. CONCLUSIONS: The study confirms that ECH elicits tissue proliferation at the contact site and corroborates the suggestion of an ECH carcinogenic effect due to hallmarks of tumoral growth, such as angiogenesis, basal membrane alterations, and loss of intercellular communication via gap junctions.

Eczema alérgico de contacto por resina epoxi / Allergic contact eczema from epoxy resin

Las resinas epoxi son plásticos muy empleados como aislantes eléctricos, en recubrimientos y como adhesivos y pinturas. Presentan un gran poder sensibilizante y son una de las principales causas de eczema alérgico de contacto, tanto en el medio laboral como fuera de éste. Presentamos el caso de un trabajador de una planta plastoquímica que manipulaba componentes de material aeronáutico en el proceso de fabricación de piezas de fuselaje. Consultó por lesiones eczematosas en los dedos, manos y antebrazos, de 2 años de evolución, en clara relación con su actividad laboral. Se realizaron pruebas epicutáneas con la batería estándar, la de plásticos y adhesivos y sus productos propios. Se objetivaron positividades a resinas epoxi (batería estándar) y a sus productos propios, entre los que se encontraban distintas láminas de fibras de vidrio y carbono impregnadas con resinas epoxi y adhesivos epoxi

Epoxy resins are plastics that are widely used as electrical insulation, in coatings, and as adhesives and paints. They have strong sensitizing power and are one of the main causes of allergic contact eczema, both in the workplace and elsewhere. We present the case of a worker at a plastics/ chemical plant, who handled aeronautical components in the process of manufacturing fuselage parts. He consulted his physician because of eczematous lesions on his fingers, hands and forearms which had developed over a two-year period and were clearly related to his work. The standard battery of skin tests was performed, along with the plastics and adhesives series and tests using the products from his workplace. Positivity was shown to epoxy resins (standard battery) and to the products from his workplace, which included different fiberglass and carbon fiber sheets impregnated with epoxy resins and epoxy adhesives

Decreased lung function associated with occupational exposure to epichlorohydrin and the modification effects of glutathione s-transferase polymorphisms.

Epichlorohydrin (1-chloro-2,3,-epoxypropane; ECH) is a strong irritant of the eyes, respiratory tract, and skin. Because the toxic effect of various chemicals can be modified by metabolic traits, in this study, we also investigated the influence of the glutathione S-transferase (GSTM1) and (GSTT1) genes on the toxic effect of ECH. In the GSTM1 null genotype workers, there is a dose-response of lung function tests (FEV1, FEV1/FVC, MMEF) for ECH exposure, but not in the GSTM1 non-null genotype workers. The ECH exposure was found to be significantly associated with a decreased FEV1 value (P = 0.09) and a decreased MMEF value (P = 0.053) after adjusting for other factors. The GSTM1 null genotype was found to be significantly associated with a decreased FEV1 value (P = 0.038), decreased FEV1/FVC value (P = 0.056), and decreased MMEF value (P = 0.012) after adjusting for other factors. This study indicates that obstructive lung abnormalities and small airway lung damage are associated with ECH exposure, and ECH workers with GSTM1 null-type are also associated with increased respiratory damage.

Genotoxic effects of ethylene oxide, propylene oxide and epichlorohydrin in humans: update review (1990-2001).

Ethylene oxide (EtO), propylene oxide (PO) and epichlorohydrin (ECH) are important industrial chemicals widely used as intermediates for various synthetic products. EtO and PO are also environmental pollutants. In this review we summarize data published during the period 1990-2001 concerning both the genotoxic and carcinogenic effects of these epoxides in humans. The use of DNA and hemoglobin adducts as biomarkers of exposure and the role of polymorphism, as well as confounding factors, are discussed. We have also included recent in vitro data comprising genotoxic effects induced by EtO, PO and ECH in mammalian cells. The uncertainties regarding cancer risk estimation still persist, in spite of the large database collected.

Increment of sister chromatid exchange frequencies (SCE) due to epichlorohydrin (ECH) in vitro treatment in human lymphocytes.

Although several studies have examined the effects on health of exposure to epichlorohydrin (ECH) through normal industrial operations and production, there is still considerable interest in its potential harmful effects on humans. The aim of the present study was to evaluate ECH effects in vitro through controlled investigations by using sister chromatid exchange (SCE), micronucleus (MN), and chromosome aberrations (CA) as the test battery. Cultures for cytogenetic tests were set up from blood samples of four healthy non-smoking and three smoking males. The experiments were performed using four different concentrations: 10(-10) M, 10(-8) M, 10(-6) M, and 10(-4) M, of ECH in DMSO. Analysis of variance showed that concentrations of ECH had significant effects on SCE/cell frequencies in the lymphocyte cultures of all donors (F=100.25, P<0.001). We were unable to find any evidence of significant increases in CA and MN frequencies in ECH-treated lymphocyte cultures with respect to the controls.