AIP Conference Proceedings 2290, 020007 (2020); https://doi.org/10.1063/5.0027353 2290, 020007
© 2020 Author(s).
Acute toxicity of the water chlorination byproduct (chloroform) in male mice
Cite as: AIP Conference Proceedings 2290, 020007 (2020); https://doi.org/10.1063/5.0027353 Published Online: 04 December 2020
Salam Hussein Ewaid, Salwan Ali Abed, and Nadhir Al-Ansari
ARTICLES YOU MAY BE INTERESTED IN
Etiology of Parkinson's disease: Toxoplasma parasite as a model
AIP Conference Proceedings 2290, 020012 (2020); https://doi.org/10.1063/5.0027539 Haematological parameters and biochemical indices in patients with chronic kidney disease before haemodialysis Al-Furat Al-Awsat Governorates/Iraq
AIP Conference Proceedings 2290, 020004 (2020); https://doi.org/10.1063/5.0027856 Immunological and physiological study of itching caused by Sarcoptes scabiei before and after treatment
AIP Conference Proceedings 2290, 020028 (2020); https://doi.org/10.1063/5.0027515
Acute Toxicity of the Water Chlorination Byproduct (Chloroform) in Male Mice
Salam Hussein Ewaid
1,a ), Salwan Ali Abed
2and Nadhir Al-Ansari
31Technical Institute of Shatra, Southern Technical University, Iraq.
2Department of Environment, College of Science, University of Al Qadisiyah, Iraq.
3Luleå University of Technology, Luleå , Sweden.
a) Corresponding author: Salwan.abed@qu.edu.iq
Abstract. Drinking water disinfection has been one of the biggest advances in public health.
However, animal studies have suggested that when tested at high concentrations, certain disinfection byproducts disinfection (DBPs) in drinking water can cause an increased incidence of cancer and reproductive effects. In this study, sixty male white mice were kept under the laboratory conditions, divided into six exposure groups and administrated with single oral dose 0, 50, 300, 700, 1000 or 1500 mg/kg BW chloroform dissolves in corn oil as a vehicle. Clinical observations and gross pathologic examination made after dosing on surviving died, and animals that were killed on the 7th day. At end of 14 days, animal weights were recorded, liver and kidneys removed, weigh and examined with the naked eye then it was fixed in 10%Neutral Buffered Formalin (NBF), transfer to 70% ethanol and included in paraffin. Tissue parts were cut 4-5 µm soiled with hematoxylin and eosin. Clinical signs observed at high doses consisted of behavioral effects, reduced body weight, livers and kidneys were congested, enlarged and their weights increased. Liver damage was characterized mainly through centrilobular necrosis and massive necrosis at higher doses. The kidney damage containing raised kidney weight, inflammation, renal cell proliferation and proximal tubular necrosis.Lethal dose causes death in 50% of exposed animals (LD50) value calculated by the probit analysis was 550 mg/kg Body Weight (BW).
Keywords: water, chloroform, mice, LD 50, liver, kidney, paraffin.
INTRODUCTION
Drinking water utilities played the main part in maintaining public health by reducing waterborne illness by liquidation, cleansing, and application of drinking water standards. Since 1974, it was discovered that chloroform was the major among four disinfection by-products (DBPs) called trihalomethanes (THMs) resulting from interaction of chlorine with water natural organic materials [1].
There is evidence on the relation between exposition to DBPs in drinking water and cancer in many epidemiological studies but proof of carcinogenicity of chlorinated drinking-water in humans is insufficient [2]. Chloroform is pure, colorless, volatile liquid with special smell and burns sweet taste. It is not hydrolyzed soluble in most organic solvents and has a chemical structure (CHCl3) [3]. It is used as a solvent and as an anesthetic, a significant amount is produced in water chlorination and bleaching of paper pulp [4]. Most of the chloroform exist in water is transfer to air because of its oscillation. It has a residence
strains of mice, liver harm basically described through early fatty infiltration and balloon cells progress to central necrosis and massive necrosis [6]. kidney also targets organs in male mice of critical strains its harm begins by hydropic degeneration and developing to necrosis of proximal tubules. Renal toxicity wasn't noticed in female mice of any strain [7]. Acute toxicity changes according to strain, sex, and vehicle.
In mice single oral dose LD50 (deadly dose causes death in 50% of exposed animals) values from 36 to 1366 mg chloroform/kg body weight, while for rats from 450 to 2000 mg chloroform/kg body weight [8].
Chloroform causes similar symptoms of poisoning in humans and animals to average oral lethal dose for humans rated 45 g but significant diversity happens portability [9]. Chloroform good absorb, metabolism and elimination through mammals after oral inhalation or dermal exposure and vastly distribute throughout organism by blood circulation, because of its lipid susceptibility
tends to stabilize
in adipose tissues and in the brain, which leads to accumulation [10]. It is basically metabolized in the liver through both oxidative and reductive paths, the main oxidation products are carbon dioxide and interactive metabolites containing phosgene. The reductive pathway produces dichloromethyl-carbene free roots. Tracks follow by cytochrome P450- based enzymatic activation and balance depend on species, tissue, dose, and oxygen tension [3].The aim of the present research is to determine target tissues for severe intoxication influences of chloroform, histopathologic responses in male mice, and to identify LD50 of chloroform.
MATERIALS AND METHODS Animals and Housing Condition
Sixty healthy male, sexually mature laboratory breed white mice (Mus musculus L.) BALB/c of an average body weight (BW) of 30 grams and 8-10 weeks old were obtained from the Iraqi Center for Cancer and Medical Genetics, animals were kept in the Department of Biology, College of Science, University of Baghdad; under the laboratory conditions (12 h light: 12h dark photoperiod), with controlled room temperature 20-25oc (October 2017), good ventilation, fed rodent diet and tap water ad Libitum, animals reared in a polypropylene cages (40×25×15 cm diameter) wrapped with a wire casings in animal house.Mice adapted to the animal facility for one week before use, randomized through weight in treatment groups and were weighed during research.
Experiment Design
Animals randomly subdivided into six groups of exposure (n = 10 mice for every group) and fitted 0, 50, 300, 700, 1000 or 1500 mg/kg body weight chloroform dissolved in corn oil (Polyoxythylated vegetable oil) like vehicle, the sixth group received only corn oil without chloroform as a control.
Chloroform dissolve in corn oil in proper condensation in order dosing volumes remain stable at 1 ml/kg and giving to animals fasted overnight utilizing early infant feeding tubes. Clinical notes made for 14 days after dosing and examine the body after death conducted on animals that died at this time. Whole remaining animals died at the end of 14 days and underwent a serious pathological examination. LD50 values studied utilizing probit analysis [11]. LD50 single oral toxicity dose of chloroform is based on the results of previous studies [12, 13, 14].
Necropsy
In every dose level of chloroform mice divided into two groups, hepatotoxicity and nephrotoxicity. The necropsy of four animals of each group was done, animal weights were recorded. Livers and kidneys removed and examined with the naked eye then stabilized in 10% neutral phosphate-buffered formalin (NBF) and sent to The Educational Laboratories of Baghdad Medical City, there it was transferred to 70%
ethanol and included in paraffin. Tissue parts were cut 4-5 µm thick and stained with hematoxylin and eosin for microscopic.
RESULTS AND DISCUSSION
After giving single doses of oral chloroform 50, 300, 700, 1000 or 1500 mg/kg body weight most of the animals give the highest doses (1000 and 1500 mg/kg BW) died within 7 days and death mostly occurred during 24 hr after dosing. At lower doses (300 and 700 mg/kg BW) animals died primarily on days 4-7 post-administration, the LD50 values calculated by were found to be 550 mg/kg BW acute poisoning data of chloroform is presented in Table 1 and Figure 1.
TABLE 1. Results of lethal doses of Chloroform for determination of LD50 in male mice (n=10).
Dose (mg/kg) Dose Log Dead % Corrected Probits
50 1.6 0 2.5 3.04
300 2.4 30 30 4.48
700 2.8 50 50 5
1000 3 70 70 5.52
1500 3.1 100 97.5 6.96
FIGURE 1. The chloroform LD50 curve in male mice.
The results of this study are generally agreed with the data of [12, 13, 14] when they found that oral LD50 values from 36 to 1366 mg chloroform/kg BW of different strains of mice [5]. Acute poisoning of chloroform is species, strain, sex and age-dependent [15]. LD50 of single oral dose in male mice changed from 120 mg/kg BW in DBA/2J mice to 490 mg/kg BW in C57BL/6J mice [13]. Clinical signs observed in this study consisted of behavioral effects at higher doses (700, 1000 mg/kg BW) like ataxia and incoordination at the highest dose (1500 mg/kg BW), anesthesia piloerection, sedation, flaccid muscle tone, ataxia, and prostration.Necropsies performed on dead animals and those killed after 7 and 14 days showed that: the body weights were reduced; livers and kidneys congested and enlarged. A significant increase in liver weights (from 1.1g to 1.5g) and the body weight was reduced (from 30g to 24g) noticed in mice at 7 and 14 days after treated with 700, 1000 or 1500 mg/kg BW chloroform. Liver damage was characterized by centrilobular necrosis and massive necrosis at the higher doses, Figure 2. The kidney damage includes raised kidney weight, inflammation, renal cell proliferation, and proximal tubular necrosis, the damage starts with hydropic degeneration at 300 mg/kg BW dose and progresses to necrosis of epithelium cells in the proximal tubules at 1500 mg/kg BW, Figure 3. Males of several mice strains oversensitive to renal tubular necrosis while females are not similarly influenced. Response to chloroform raised through mice age strains C3H, C3Hf, A and HR and C57BL, C57L, C57BR/cd, and ST susceptible to exposure
R² = 0.8642
0 1 2 3 4 5 6 7 8
0 0.5
1 1.5
2 2.5
3 3.5
Probits
Log of dose
Many researches refer male rather female mice sensitive to chloroform caused by nephrotoxicity [20, 21, 22].
FIGURE 2. (a, b) Normal liver from a male control mouse. (c, d) Treated mouse after administering 1500 mg chloroform/kg BW shows prevalent centrilobular hepatic necrosis and expansion of middle sinus through inflammatory
cells (dark zones).
FIGURE 3. (a, b) Normal kidney cortex and whole kidney from a male control mouse.
(c, d) The kidney cortex of male mouse treated 1500 mg chloroform/kg shows nearby proximal tubules, necrotic and lined through degenerated epithelium necrotic cells scattered into the lumen. (e) Treated kidney medulla shows the
necrotic zones (dark).
CONCLUSIONS
Drinking water disinfection byproducts became a problem in public health. Animal studies have suggested that DBPs can cause an increased incidence of cancer and reproductive effects. Several epidemiological studies have suggested that chlorinated drinking water could be associated with increased incidences of bladder, rectal and colon cancer and harmful impacts on reproduction. Currently there are limited data on the next generation of drinking water standards to make sound scientific judgements.This study was a contribution to the attempts aimed at determine the target tissues for intoxication influences of chloroform, histopathologic responses in male mice, and to identify LD50 of chloroform.
REFERENCES
1. WHO (World Health Organization) (2005). Trihalomethanes in Drinking-water, Background
3. Ewaid, S. H., Rabee, A. M., & Al-Naseri, S. K. (2018). Carcinogenic Risk Assessment of Trihalomethanes in Major Drinking Water Sources of Baghdad City. Water resources, 45(5), 803 - 812.
4. Liu ,S; Yao, Y; Lu, S; Aldous, K; Ding X; Me,i C; Gu ,J. (2013). The role of renal proximal tubule P450 enzymes in chloroform-induced nephrotoxicity: utility of renal specific P450 reductase knockout mouse models. . Toxicol Appl Pharmacol, 230-7.
5. International Programme on Chemical Safety (IPCS) 2004. Chloroform. Concise International Chemical Assessment Document 58. 2004, WHO: Geneva.
6. Morcos, N. Y., Diab, M. R., Allah, A. A., & HIA, I. (2015). Acute health effects of chloroform and xylene in mice.
7. Fang, C; Behr, M; Xie, F; Lu, S; Doret, M; Luo, H; Yang, W; Aldous, K; Ding, X; Gu, J; (2008).
Mechanism of chloroform-induced renal toxicity: non-involvement of hepatic cytochrome P450- dependent metabolism. Toxicol Appl Pharmacol, 48-55.
8. Larson, JL, Wolf, DC & Butterworth, BE. (1993). Acute hepatotoxic and nephrotoxic effects of chloroform in male F-344 rats and female B6C3F1 mice. Fundam Appl Toxicol 20:302-315.
9. Agency for Toxic Substances and Disease Registry (ATSDR) (1997). Toxicological Profile for Chloroform. US Department of Health and Human Services: Atlanta, US.
10. Hill, R.N. (1977). Differential toxicity of chloroform in the mouse. Ann NY Acad Sci 298: 170 - 176. Cited in (Health Canada, 2009) Trihalomethanes, Guidelines for Canadian Drinking Water Quality. Federal-Provincial-Territorial Committee on Health and the Environment Ottawa, Ontario.
11. Finney, D. J., Ed. (1952). Probit Analysis. Cambridge, England, Cambridge University Press.
Cited in (Larson et al., 1993).
12. Bowman, F.J., Borzelleca J.F. and Munson, A.E. (1978). The toxicity of some halomethanes in mice. Toxicology and Applied Pharmacology, 44:213–215.
13. Hill, R.N., Clemens, T.L., Liu, D.K. & Johnson, W.D.age-dependent (1975). Genetic control of chloroform toxicity in mice. Science, 190, 159–161. Cited in (Health Canada, 2009).
14. Pericin, C. and Thomann, P. (1979). Comparison of the acute toxicity of clioquinol, histamine and chloroform in different strains of mice. Arch Toxicol 2 (Suppl):371-3.
15. Sasso ,AF; Schlosser ,PM; Kedderis, GL; Genter ,MB; Snawder, JE; Li ,Z; Rieth, S; Lipscomb ,JC (2013). Application of an updated physiologically based pharmacokinetic model for chloroform to evaluate CYP2E1-mediated renal toxicity in rats and mice. Toxicol Sci. , 260-74.
16. Deringer, M.K., Dunn, T.B., Heston, W.E. (1953). Results of exposure of strain C3H mice to chloroform. Proc Soc Exp Biol Med 83: 474–479, cited in U.S. Environmental Protection Agency (U.S. EPA, 2001) Toxicological Review of Chloroform.
17. Larson, J.L., Wolf, D.C. & Butterworth, B.E. (1994). Lack of chloroform-induced DNA repair in vitro and in vivo in hepatocytes of female B6C3F1 mice. Environ Mol Mutagen 23:132-136.
18. Larson, J.L., Wolf, D.C. & Butterworth, B.E. 1994b. Induced cytotoxicity and cell proliferation in the hepatocarcinogenicity of chloroform in female B6C3F1 mice: Comparison of administration by gavage in corn oil. Fundam Appl Toxicol 22:90-102.
19. Riederer, A. M., Dhingra, R., Blount, B. C., & Steenland, K. (2014). Predictors of blood trihalomethane concentrations in NHANES 1999–2006. Environmental health perspectives, 122(7), 695-702.
20. Nagano K, Kano H, Arito H, Yamamoto S, Matsushima T. (2006). Enhancement of renal carcinogenicity by combined inhalation and oral exposures to chloroform in male rats. J Toxicol Environ Health A. , 1827-42.
21. Templin, M.V; Constan, A.A.; Wolf, D.C. (1998). Patterns of chloroform-induced regenerative cell proliferation in BDF1 mice correlate with organ specificity and dose-response of tumor formation. Carcinogenesis 19:187-193.
22. Take, M; Takeuchi, T; Haresaku, M; Matsumoto, M; Nagano, K; Yamamoto, S; Takamura-Enya, T; Fukushima, S (2010). Estimation of chloroform inhalation dose by other routes based on the relationship of area under the blood concentration-time curve (AUC)-inhalation dose to chloroform distribution in the blood of rats. . J Environ Sci Health A Tox Hazard Subst Environ Eng, 253-61.
