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Effects of methanolic leaf extract of salacia senegalensis (Lam) DC on some biochemical and haematological indices in albino mice
Abstracts / Toxicology Letters 238S (2015) S56–S383
professionals, safety representatives and authority employees. REACH has increased the number of available exposure limits for the occupational setting. Currently more than 3000 individual substances have wDNELs (www.dguv.de/ifa/Gefahrstoffdatenbanken/ GESTIS-DNEL-Datenbank/index-2.jsp, 2015-03-15). This can be compared with the number of substances on member states’ lists of OELs that cover some 100–800 individual substances (Ding et al., 2011, Regul. Toxicol. Pharmacol. 61: 296–309). Swedish OELs and wDNELs were available for 235 substances. Although on average these exposure benchmarks were at the same level the variation was huge, ranging from 450 times higher to 230 times lower than the corresponding OEL. Nearly one fifth of the wDNELs were ≥2 times higher and one third ≥2 times lower than the OEL. Age of the OEL did not correlate with the wDNEL/OEL ratios. The interviews showed that many of our informants were not aware of the concept of wDNELs, informants who were aware of DNELs found their relationship to OELs unclear. This group tended to either disregard wDNELs completely in their practice or at most view them as something to compare OELs with. Only authority informants connected wDNELs to the building of exposure scenarios. Despite the concerns about the quality of wDNELs the large effort in establishing a first exposure benchmark for the many substances lacking OELs is a valuable outcome of REACH. However, one implication of the low awareness of wDNELs is that this resource most likely is underutilized as a source of risk information. http://dx.doi.org/10.1016/j.toxlet.2015.08.441
P04-006 Nickel oxide particles cause hydrolysis of alpha-1 antitrypsin, key protease inhibitor of human blood ˛ N. Wezynfeld ∗ , W. Bal, T. Fraczyk Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland The purpose: Nickel is a very common industrial metal. Coins, jewellery, mobile phone cases, and kitchenware are typical examples of everyday use objects leading to contact of human skin with nickel. On the other hand, nickel is a human toxin, causing contact allergy, lung cancer, and acute toxicity after ingestion. Lungs are among the most exposed organs to nickel, under such circumstances as industrial exposure to nickel containing dusts and use of electronic cigarettes. Nickel can also cause metal-dependent hydrolysis of peptides or proteins in Aaa-Ser/Thr-Xaa-His-Zaa sequences. This specific reaction, depending on the location of susceptible sequence(s) in their structure, can lead to a partial or full loss of the protein function. One of proteins possessing sequences susceptible to Ni(II)-dependent hydrolysis is human alpha-1 antitrypsin (AAT). It is an important serine protease inhibitor. Deficiency of this protein results in serious lung diseases, because insufficient inhibition of elastase leads to breakdown of elastin, causing damage of connective tissue of the lungs. The aim of the study was to carry out hydrolysis of AAT in the presence of slightly soluble nickel containing particles, which are the major forms of nickel entering into the respiratory tract. Methods: Samples of AAT and slightly soluble particulate nickel compounds (black and green nickel oxides, nickel oxide nanoparticles) were incubated at 37 ◦ C. Aliquots were periodically collected from reaction mixtures, frozen in liquid nitrogen and stored at −20 ◦ C until analyzed using HPLC (C18 column), SDS-PAGE, ESI-MS and MALDI-MS.
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Results: AAT hydrolysis was found to occur in the presence of slightly soluble particulate nickel compounds at both susceptible sequences of this protein, close to its N- and C-termini. Such cleavage leads to inactivation of this inhibitor, as shown by us previously. Nickel oxide nanoparticles were found to be the most effective nickel ion source for this reaction. Conclusions: Ni(II) dependent hydrolysis can contribute to AATrelated pathologies and can be an important aspect of nickel toxicity in human respiratory tract. http://dx.doi.org/10.1016/j.toxlet.2015.08.442 P05 – Clinical Toxicology P05-001 Effectiveness of multiple-dose activated charcoal in digoxin overdose: A case report R. Ponampalam Singapore General Hospital, Emergency Medicine, Singapore, Singapore Introduction: Multiple-dose activated charcoal (MDAC) is thought to enhance the elimination of digoxin and may be an effective and inexpensive treatment option for digoxin toxicity. Case report: A 35-year-old female with a history of depression ingested approximately 40 tablets of digoxin (62.5 mcg tablets, total 2.5 mg) 9 h prior to presentation. The patient complained of epigastric discomfort and nausea upon arrival to emergency department (ED). Electrocardiogram (ECG) revealed sinus bradycardia with heart rate (HR) of 30 beats/min and blood pressure (BP) 90/51 mmHg. The serum digoxin level 9 h post-ingestion was 8.9 g/L and potassium level was 4.3 mmol/L. The antidote was withheld but the patient was given 50 g of activated charcoal (AC) every 2 h for 4 doses and poly ethylene glycol (PEG) solution after charcoal administration. The digoxin level decreased from 8.9 to 3.6 g/L (59.6% reduction) over 19 h. The expected level without MDAC based on elimination half-life of 42 h would be 6.9 g/L (22.5% reduction). Discussion: MDAC is effective in enhancing the elimination of digoxin in overdosed patients. As digoxin-specific antibodies have serious adverse effects and are expensive, MDAC should be considered for patients with digoxin poisoning who are symptomatic but with no evidence of serious toxicity (i.e. serum potassium < 5.0 mmol/dL, serum digoxin level < 10.0 g/L, ingestion of less than 10 mg of digoxin or symptomatic arrhythmias responsive to advanced cardiac life support guidelines). http://dx.doi.org/10.1016/j.toxlet.2015.08.444
P05-002 Effects of methanolic leaf extract of salacia senegalensis (Lam) DC on some biochemical and haematological indices in albino mice O. Adumanya Imo State Polytechnic, Science Lab. Tech, Owerri, Nigeria Salacia senegalensis – a medicinal plant with an established antimalarial activity was assayed for its methanolic leaf extract effects on some biochemical and haematological parameters using Swiss albino mice. Biochemical parameters assayed were serum alanine aminotransferase (ALT), aspartate aminotransferase (AST)
S142
Abstracts / Toxicology Letters 238S (2015) S56–S383
and total protein, while haemoglobin (Hb), platelets (Plat), total white blood cell (WBC), lymphocyte (Lymp) and neutrophil (Neut) levels were the haematological indices measured. The albino mice (six per group) weighing 18–22 g used for the haematology and biochemical studies were grouped into A (control) given 5 mL normal saline/kg/body weight per day, B (given 1000 mg), C (given 1200 mg) and D given 1400 mg extracts respectively/kg body weight per day for one week. The haematological results showed that the extract produced a dose-dependent increase in haemoglobin concentration in all the animal groups which were significantly higher than that of mice in the control group (P < 0.05). The platelets levels were significantly reduced compared to the control group, while the white blood cells, lymphocytes and neutrophils levels of the animals in groups B–D were significantly increased compared to the control (P < 0.05). These indicate the haemopoietic and the immune-stimulatory properties of the extract. The biochemical results showed that the serum aspartate and alanine aminotransferase levels were significantly increased compared to the control (P < 0.05), while a decrease was observed in protein levels compared to the control (P > 0.05). The weights of the animals were not significantly altered compared to the control group. Photomicrographs of the extract-treated albino mice livers showed a moderate inflammatory response at the extract doses used. The results suggest a mild hepatotoxic effect after one week (prolonged) treatment with the methanol extract of Salacia senegalensis leaves at various doses used. http://dx.doi.org/10.1016/j.toxlet.2015.08.445
P05-003 Do novel psychoactive substances (NPS) produce similar pharmacological effects to their classified chemical derivatives or are they responsible for unpredictable clinical outcomes A. Mc Callum Royal Sussex County Hospital, Medicine, Brighton, United Kingdom Background: The designer drug market has expanded rapidly with over 164 new NPS emerging. The constant innovation to replace classified NPS has led to limited knowledge of both the pharmacodynamics and pharmacokinetics of the newer substances. This has been extremely challenging for doctors managing patients with unpredictable symptoms secondary to acute toxicity. Methods: A retrospective case series was carried out over a 20 week period in the emergency department at The Royal Sussex County hospital. A sample size of 49 presentations with primary drug intoxication was included. Clinical parameters were recorded from pre-hospital and hospital data. Cluster analysis was used as a statistical method to identify the correlation in clinical symptoms and drug groups. Results: During a 20 week period, 10 classes of drugs were identified, with the most common drug presentations being: MDMA, cocaine, ketamine and 2CB. A dendrogram identified four distinct natural groupings. Cluster one showed a wide variety of drug groups which had a similar mean GCS. The strongest linkages were between cocaine, ketamine, herbal haze and cannabis. Cluster two consisted of drug groups MDMA, 2CB, benzofury and mephedrone and identified a significant tachycardia amongst subjects. Cluster three and four identified standalone cases of severe MDMA toxicity with tachycardia and hypertension. Conclusion: Although there were some similarities in drug classes within the clusters, many drugs did not present in a classical way and showed unusual groupings. Clinical presentation is
clearly affected by both drug and human factors including: drug purity, adulterants, polydrug use, stereoisomers within compounds and inter-individual variability. Therefore it is extremely difficult to distinguish between drug groups based on clinical presentation. As very little is known about the interaction and metabolites of NPS, symptomatic management is currently the optimal treatment in acute presentations. http://dx.doi.org/10.1016/j.toxlet.2015.08.446
P05-004 The interaction between the MRP1 G1666A and MRP2 G4544A polymorphisms on the blood and urinary arsenic levels D. Kaya-Akyüzlü 1,∗ , Z. Kayaaltı 1 , F. Özdemir 1 , V.A. Türksoy 1 , E. Tutkun 2 , T. Söylemezo˘glu 1 1 Ankara University, Institute of Forensic Sciences, Forensic Toxicology, Ankara, Turkey 2 Ankara Occupational Diseases Hospital, Ankara, Turkey
Question: Inorganic arsenic (arsenate-AsV and arsenite-AsIII) is mainly biomethylated to various metabolites by a sequence of two distinct enzymatically catalyzed reactions in human liver and excreted into urine and feces. Human Multidrug Resistance Protein 1 (MRP1) and the related protein MRP2 are ATP-Binding Cassette (ABC) transporter proteins that protect tissues from arsenic accumulation and resulting toxicity through the cellular efflux of arsenic-glutathione conjugates. In polarized cells, MRP1 (ABCC1) localizes to basolateral plasma membrane, whereas MRP2 (ABCC2) is expressed on the apical membrane and functions in the biliary transport of arsenic. Taken together, we hypothesized that single nucleotide polymorphisms in ABCC1 and ABCC2 genes may influence the excretion of inorganic arsenic. Methods: The study population comprised 95 Turkish smelter workers. MRP1 G1666A and MRP2 G4544A polymorphisms were investigated by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. Blood and urinary arsenic concentrations were measured by Graphite Furnace Atomic Absorption Spectroscopy (GFAAS) with Zeeman correction. Results: The mean blood and urinary arsenic levels were 21.60 ± 12.28 g/L and 5.58 ± 4.37 g/L, respectively. The genotype frequencies were found as 12.6% homozygote typical (GG), 41.1% heterozygote (GA) and 46.3% homozygote atypical (AA) for MRP1 G1666A and 84.2% homozygote typical (GG) and 15.8% heterozygote (GA) for MRP2 G4544A. There was a significant association between MRP1 G1666A and MRP2 G4544A polymorphisms and urinary arsenic levels (p 0.05). Also, to understand the effect of the combination of MRP1 G1666A and MRP2 G4544A polymorphisms on blood and urinary arsenic levels, subjects were divided into four groups according to genotypes: MRP1A− /MRP2A− , MRP1A− /MRP2A+ , MRP1A+ /MRP2A− and MRP1A+ /MRP2A+ . Highly significant association was detected between MRP1A− /MRP2A+ genotypes and urinary arsenic levels (p = 0.001). Individuals with the MRP1A+ /MRP2A+ had higher blood arsenic level (27.81 ± 13.85 g/L) than those with MRP1A− /MRP2A− (15.60 ± 8.11 g/L), MRP1A− /MRP2A+ (19.80 ± 19.07 g/L) and MRP1A+ /MRP2A− (21.35 ± 12.06 g/L) genotypes (p = 0.121), but this result was not statistically significant. Conclusion: The interaction between the MRP1 G1666A and MRP2 G4544A polymorphisms were associated with interindividual variations in urinary and blood arsenic levels. http://dx.doi.org/10.1016/j.toxlet.2015.08.447
professionals, safety representatives and authority employees. REACH has increased the number of available exposure limits for the occupational setting. Currently more than 3000 individual substances have wDNELs (www.dguv.de/ifa/Gefahrstoffdatenbanken/ GESTIS-DNEL-Datenbank/index-2.jsp, 2015-03-15). This can be compared with the number of substances on member states’ lists of OELs that cover some 100–800 individual substances (Ding et al., 2011, Regul. Toxicol. Pharmacol. 61: 296–309). Swedish OELs and wDNELs were available for 235 substances. Although on average these exposure benchmarks were at the same level the variation was huge, ranging from 450 times higher to 230 times lower than the corresponding OEL. Nearly one fifth of the wDNELs were ≥2 times higher and one third ≥2 times lower than the OEL. Age of the OEL did not correlate with the wDNEL/OEL ratios. The interviews showed that many of our informants were not aware of the concept of wDNELs, informants who were aware of DNELs found their relationship to OELs unclear. This group tended to either disregard wDNELs completely in their practice or at most view them as something to compare OELs with. Only authority informants connected wDNELs to the building of exposure scenarios. Despite the concerns about the quality of wDNELs the large effort in establishing a first exposure benchmark for the many substances lacking OELs is a valuable outcome of REACH. However, one implication of the low awareness of wDNELs is that this resource most likely is underutilized as a source of risk information. http://dx.doi.org/10.1016/j.toxlet.2015.08.441
P04-006 Nickel oxide particles cause hydrolysis of alpha-1 antitrypsin, key protease inhibitor of human blood ˛ N. Wezynfeld ∗ , W. Bal, T. Fraczyk Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland The purpose: Nickel is a very common industrial metal. Coins, jewellery, mobile phone cases, and kitchenware are typical examples of everyday use objects leading to contact of human skin with nickel. On the other hand, nickel is a human toxin, causing contact allergy, lung cancer, and acute toxicity after ingestion. Lungs are among the most exposed organs to nickel, under such circumstances as industrial exposure to nickel containing dusts and use of electronic cigarettes. Nickel can also cause metal-dependent hydrolysis of peptides or proteins in Aaa-Ser/Thr-Xaa-His-Zaa sequences. This specific reaction, depending on the location of susceptible sequence(s) in their structure, can lead to a partial or full loss of the protein function. One of proteins possessing sequences susceptible to Ni(II)-dependent hydrolysis is human alpha-1 antitrypsin (AAT). It is an important serine protease inhibitor. Deficiency of this protein results in serious lung diseases, because insufficient inhibition of elastase leads to breakdown of elastin, causing damage of connective tissue of the lungs. The aim of the study was to carry out hydrolysis of AAT in the presence of slightly soluble nickel containing particles, which are the major forms of nickel entering into the respiratory tract. Methods: Samples of AAT and slightly soluble particulate nickel compounds (black and green nickel oxides, nickel oxide nanoparticles) were incubated at 37 ◦ C. Aliquots were periodically collected from reaction mixtures, frozen in liquid nitrogen and stored at −20 ◦ C until analyzed using HPLC (C18 column), SDS-PAGE, ESI-MS and MALDI-MS.
S141
Results: AAT hydrolysis was found to occur in the presence of slightly soluble particulate nickel compounds at both susceptible sequences of this protein, close to its N- and C-termini. Such cleavage leads to inactivation of this inhibitor, as shown by us previously. Nickel oxide nanoparticles were found to be the most effective nickel ion source for this reaction. Conclusions: Ni(II) dependent hydrolysis can contribute to AATrelated pathologies and can be an important aspect of nickel toxicity in human respiratory tract. http://dx.doi.org/10.1016/j.toxlet.2015.08.442 P05 – Clinical Toxicology P05-001 Effectiveness of multiple-dose activated charcoal in digoxin overdose: A case report R. Ponampalam Singapore General Hospital, Emergency Medicine, Singapore, Singapore Introduction: Multiple-dose activated charcoal (MDAC) is thought to enhance the elimination of digoxin and may be an effective and inexpensive treatment option for digoxin toxicity. Case report: A 35-year-old female with a history of depression ingested approximately 40 tablets of digoxin (62.5 mcg tablets, total 2.5 mg) 9 h prior to presentation. The patient complained of epigastric discomfort and nausea upon arrival to emergency department (ED). Electrocardiogram (ECG) revealed sinus bradycardia with heart rate (HR) of 30 beats/min and blood pressure (BP) 90/51 mmHg. The serum digoxin level 9 h post-ingestion was 8.9 g/L and potassium level was 4.3 mmol/L. The antidote was withheld but the patient was given 50 g of activated charcoal (AC) every 2 h for 4 doses and poly ethylene glycol (PEG) solution after charcoal administration. The digoxin level decreased from 8.9 to 3.6 g/L (59.6% reduction) over 19 h. The expected level without MDAC based on elimination half-life of 42 h would be 6.9 g/L (22.5% reduction). Discussion: MDAC is effective in enhancing the elimination of digoxin in overdosed patients. As digoxin-specific antibodies have serious adverse effects and are expensive, MDAC should be considered for patients with digoxin poisoning who are symptomatic but with no evidence of serious toxicity (i.e. serum potassium < 5.0 mmol/dL, serum digoxin level < 10.0 g/L, ingestion of less than 10 mg of digoxin or symptomatic arrhythmias responsive to advanced cardiac life support guidelines). http://dx.doi.org/10.1016/j.toxlet.2015.08.444
P05-002 Effects of methanolic leaf extract of salacia senegalensis (Lam) DC on some biochemical and haematological indices in albino mice O. Adumanya Imo State Polytechnic, Science Lab. Tech, Owerri, Nigeria Salacia senegalensis – a medicinal plant with an established antimalarial activity was assayed for its methanolic leaf extract effects on some biochemical and haematological parameters using Swiss albino mice. Biochemical parameters assayed were serum alanine aminotransferase (ALT), aspartate aminotransferase (AST)
S142
Abstracts / Toxicology Letters 238S (2015) S56–S383
and total protein, while haemoglobin (Hb), platelets (Plat), total white blood cell (WBC), lymphocyte (Lymp) and neutrophil (Neut) levels were the haematological indices measured. The albino mice (six per group) weighing 18–22 g used for the haematology and biochemical studies were grouped into A (control) given 5 mL normal saline/kg/body weight per day, B (given 1000 mg), C (given 1200 mg) and D given 1400 mg extracts respectively/kg body weight per day for one week. The haematological results showed that the extract produced a dose-dependent increase in haemoglobin concentration in all the animal groups which were significantly higher than that of mice in the control group (P < 0.05). The platelets levels were significantly reduced compared to the control group, while the white blood cells, lymphocytes and neutrophils levels of the animals in groups B–D were significantly increased compared to the control (P < 0.05). These indicate the haemopoietic and the immune-stimulatory properties of the extract. The biochemical results showed that the serum aspartate and alanine aminotransferase levels were significantly increased compared to the control (P < 0.05), while a decrease was observed in protein levels compared to the control (P > 0.05). The weights of the animals were not significantly altered compared to the control group. Photomicrographs of the extract-treated albino mice livers showed a moderate inflammatory response at the extract doses used. The results suggest a mild hepatotoxic effect after one week (prolonged) treatment with the methanol extract of Salacia senegalensis leaves at various doses used. http://dx.doi.org/10.1016/j.toxlet.2015.08.445
P05-003 Do novel psychoactive substances (NPS) produce similar pharmacological effects to their classified chemical derivatives or are they responsible for unpredictable clinical outcomes A. Mc Callum Royal Sussex County Hospital, Medicine, Brighton, United Kingdom Background: The designer drug market has expanded rapidly with over 164 new NPS emerging. The constant innovation to replace classified NPS has led to limited knowledge of both the pharmacodynamics and pharmacokinetics of the newer substances. This has been extremely challenging for doctors managing patients with unpredictable symptoms secondary to acute toxicity. Methods: A retrospective case series was carried out over a 20 week period in the emergency department at The Royal Sussex County hospital. A sample size of 49 presentations with primary drug intoxication was included. Clinical parameters were recorded from pre-hospital and hospital data. Cluster analysis was used as a statistical method to identify the correlation in clinical symptoms and drug groups. Results: During a 20 week period, 10 classes of drugs were identified, with the most common drug presentations being: MDMA, cocaine, ketamine and 2CB. A dendrogram identified four distinct natural groupings. Cluster one showed a wide variety of drug groups which had a similar mean GCS. The strongest linkages were between cocaine, ketamine, herbal haze and cannabis. Cluster two consisted of drug groups MDMA, 2CB, benzofury and mephedrone and identified a significant tachycardia amongst subjects. Cluster three and four identified standalone cases of severe MDMA toxicity with tachycardia and hypertension. Conclusion: Although there were some similarities in drug classes within the clusters, many drugs did not present in a classical way and showed unusual groupings. Clinical presentation is
clearly affected by both drug and human factors including: drug purity, adulterants, polydrug use, stereoisomers within compounds and inter-individual variability. Therefore it is extremely difficult to distinguish between drug groups based on clinical presentation. As very little is known about the interaction and metabolites of NPS, symptomatic management is currently the optimal treatment in acute presentations. http://dx.doi.org/10.1016/j.toxlet.2015.08.446
P05-004 The interaction between the MRP1 G1666A and MRP2 G4544A polymorphisms on the blood and urinary arsenic levels D. Kaya-Akyüzlü 1,∗ , Z. Kayaaltı 1 , F. Özdemir 1 , V.A. Türksoy 1 , E. Tutkun 2 , T. Söylemezo˘glu 1 1 Ankara University, Institute of Forensic Sciences, Forensic Toxicology, Ankara, Turkey 2 Ankara Occupational Diseases Hospital, Ankara, Turkey
Question: Inorganic arsenic (arsenate-AsV and arsenite-AsIII) is mainly biomethylated to various metabolites by a sequence of two distinct enzymatically catalyzed reactions in human liver and excreted into urine and feces. Human Multidrug Resistance Protein 1 (MRP1) and the related protein MRP2 are ATP-Binding Cassette (ABC) transporter proteins that protect tissues from arsenic accumulation and resulting toxicity through the cellular efflux of arsenic-glutathione conjugates. In polarized cells, MRP1 (ABCC1) localizes to basolateral plasma membrane, whereas MRP2 (ABCC2) is expressed on the apical membrane and functions in the biliary transport of arsenic. Taken together, we hypothesized that single nucleotide polymorphisms in ABCC1 and ABCC2 genes may influence the excretion of inorganic arsenic. Methods: The study population comprised 95 Turkish smelter workers. MRP1 G1666A and MRP2 G4544A polymorphisms were investigated by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. Blood and urinary arsenic concentrations were measured by Graphite Furnace Atomic Absorption Spectroscopy (GFAAS) with Zeeman correction. Results: The mean blood and urinary arsenic levels were 21.60 ± 12.28 g/L and 5.58 ± 4.37 g/L, respectively. The genotype frequencies were found as 12.6% homozygote typical (GG), 41.1% heterozygote (GA) and 46.3% homozygote atypical (AA) for MRP1 G1666A and 84.2% homozygote typical (GG) and 15.8% heterozygote (GA) for MRP2 G4544A. There was a significant association between MRP1 G1666A and MRP2 G4544A polymorphisms and urinary arsenic levels (p 0.05). Also, to understand the effect of the combination of MRP1 G1666A and MRP2 G4544A polymorphisms on blood and urinary arsenic levels, subjects were divided into four groups according to genotypes: MRP1A− /MRP2A− , MRP1A− /MRP2A+ , MRP1A+ /MRP2A− and MRP1A+ /MRP2A+ . Highly significant association was detected between MRP1A− /MRP2A+ genotypes and urinary arsenic levels (p = 0.001). Individuals with the MRP1A+ /MRP2A+ had higher blood arsenic level (27.81 ± 13.85 g/L) than those with MRP1A− /MRP2A− (15.60 ± 8.11 g/L), MRP1A− /MRP2A+ (19.80 ± 19.07 g/L) and MRP1A+ /MRP2A− (21.35 ± 12.06 g/L) genotypes (p = 0.121), but this result was not statistically significant. Conclusion: The interaction between the MRP1 G1666A and MRP2 G4544A polymorphisms were associated with interindividual variations in urinary and blood arsenic levels. http://dx.doi.org/10.1016/j.toxlet.2015.08.447