Safety evaluation of Se-methylselenocysteine as nutritional selenium supplement: Acute toxicity, genotoxicity and subchronic toxicity

Regulatory Toxicology and Pharmacology 70 (2014) 720–727

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Safety evaluation of Se-methylselenocysteine as nutritional selenium supplement: Acute toxicity, genotoxicity and subchronic toxicity Hui Yang, Xudong Jia ⇑ Key Laboratory of Food Safety Risk Assessment of Ministry of Health, China National Center for Food Safety Risk Assessment, Beijing 100021, China

a r t i c l e

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Article history: Received 7 August 2014 Available online 31 October 2014 Keywords: Selenium Se-methylselenocysteine Safety evaluation Food supplements Benchmark dose

a b s t r a c t The significant toxicity of selenium emphasizes the need to assess the health risk of various selenocompounds as nutritional supplements. Se-methylselenocysteine (SeMC) was recently reported to be more bioactive but the toxicological effects have not been sufficiently characterized. This study aimed to evaluate the safety of SeMC and provide the Acceptable Daily Intake (ADI) for its use in human diet. Our results demonstrated that SeMC, with the Median Lethal Dose (LD50) of 12.6 and 9.26 mg/kg BW in female and male mice, was of high potent of health hazard under acute oral exposure, but a battery of tests including Ames test, micronucleus assay and mouse sperm malformation assay suggested that SeMC was not genotoxic. The repeated dose study indicated little systemic toxicity of SeMC at supernutritional levels (0.5, 0.7, 0.9 mg/kg BW/day) after 90-day oral exposure. Importantly, the 95% lower confidence value of Benchmark Dose (BMDL) was estimated as 0.34 mg/kg BW/day according to the elevated relative liver weight. The ADI for human was established at 3.4 lg/kg BW/day. The results suggested greater safety of SeMC as a nutritional selenium supplement, but health risk needs to be further evaluated when SeMC is applied beyond this level to achieve cancer chemoprevention. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction As an essential micronutrient for human, selenium has been studied for nearly sixty years since its biological function was discovered in 1957 (Schwarz, 1976). It has been proven that selenium is a critical component of proteins required for various biological functions, such as antioxidant defense, reduction of inflammation, thyroid hormone production, fertility (Brown and Arthur, 2001; Whanger, 2002). Due to its essentiality for mammalian life, selenium has been utilized as a feed supplement for livestock especially in geographical areas that are naturally low in selenium. In addition, selenium is also suggested to be used for nutritional fortification in human food to prevent or rectify selenium deficiency (e.g. Keshan disease in China) (Alfthan et al., 2014; Chen, 2012; Kieliszek and Blazejak, 2013). However, selenium may induce toxic reactions at levels several times that normally ingested in the human diet (Thomson, 2004; Yang and Xia, 1995). Epidemiological studies and case reports have shown that chronic dietary exposure to selenium compounds (500 lg selenium per day) is associated with several adverse health effects in humans such as disruption of endocrine function, impartment of ⇑ Corresponding author at: 7 Panjiayuan Nanli, Beijing 100021, China. E-mail address: [email protected] (X. Jia). http://dx.doi.org/10.1016/j.yrtph.2014.10.014 0273-2300/Ó 2014 Elsevier Inc. All rights reserved.

immunity, hepatoxicity and amyotrophic lateral sclerosis (Bratter and Negretti de Bratter, 1996; Vinceti et al., 2001). Currently, the tolerable upper intake level (UL) of selenium has been set at 400 lg/day, which is about 7 times the recommended dietary allowance (RDA) in the United States (55 lg/day) (FNB, 2000). However, it was believed that intake of selenium above normal nutritional range can confer more health benefits. For example, selenium has been previously suggested possessing anticancer activity at supernutritional levels (e.g. 200 lg/day) (Thomson, 2004). Thus, the biological study on selenium in the last century has been marked by the controversial balance between efficacy and safety. It has been proven that the toxicity of selenium is dependent on the chemical speciation (WHO/IPCS, 1987). Elemental selenium and most metallic selenides have relatively less toxicity because of the low bioavailability, which on the other hand limited their utility in feed and food nutritional supplementation. By contrast, selenates, selenites and organoselenium compounds, such as selenomethionine (SeMet), selenocysteine and methylselenocysteine are widely used as nutritional selenium source but they are all toxic in large doses. In general, SeMet and SeMet enriched yeast are more effective in increasing body selenium levels and less toxic than inorganic selenium. The reason may lie on the nonspecific incorporation of selenomethionine into proteins as the

H. Yang, X. Jia / Regulatory Toxicology and Pharmacology 70 (2014) 720–727

amino acid methionine and further providing reversible selenium storage in organs and tissues (Schrauzer, 2003). However, the excessive incorporation can also lead to structural malformation or loss of enzymatic activity in some sulfur-containing proteins due to the replacement of sulfur in sulfhydryl groups or thiols (critical for disulfide bond formation) with selenium (Stadtman, 1990). Se-methylselenocysteine (SeMC), another naturally occurring organoselenium compound firstly identified in Astragalus bisulcatus (Trelease et al., 1960) and later found from many other plants (Freeman et al., 2012; Lyi et al., 2005), was reported to be less toxic and more bioactive than inorganic or other organic selenium compounds (Hoefig et al., 2011). Because SeMC is not readily incorporated into proteins and accumulates in a free pool after ingestion, it has been considered a better form of nutritional selenium supplements (Neuhierl and Bock, 1996). In addition, recent studies indicated that SeMC conferred remarkable protection against breast cancer (El-Bayoumy and Sinha, 2004; Ip et al., 2000; Unni et al., 2005; Zhang and Zarbl, 2008), prostate cancer (Sinha et al., 2014; Zhang et al., 2010) and colorectal carcinoma (Cao et al., 2014). SeMC can be a similar or better selenium source than SeMet and supplies methylselenol, an active metabolite recognized essential for the anticancer effect (Ip et al., 2002; Zhan et al., 2013), much more efficiently in organs than SeMet (Suzuki et al., 2006). It is promising that SeMC can be developed as a pharmaceutical drug that can be used in chemoprevention and clinical intervention of human cancers. However, a common concern in the uses of SeMC either for nutritional supplementation or for cancer chemoprevention is the safety risk rising from significant toxicity of selenium, and this has not been well elucidated yet. In this study, we aimed to evaluate the toxicity of SeMC through multiple in vitro and in vivo experiments. Median Lethal Dose (LD50) was established for the classification of acute oral toxicity; bacterial reverse mutation assay, mouse bone marrow micronucleus assay and mouse sperm malformation assay were performed to determine the genotoxicity of SeMC; a 90-day feeding experiment was carried out to investigate the subchronic oral toxicity, and the benchmark dose (BMD) approach was applied to estimate a point of departure for the hazard risk assessment of SeMC. 2. Materials and methods All aspects in this project involving animal care, use, and welfare were performed in compliance with the Food and Drug Administration (FDA) principles of GLP and in accordance with the FDA Guidance for Industry and Other Stakeholders, ‘‘Toxicological Principles for the Safety Assessment of Food Ingredients Redbook 2000’’ (FDA, 2000). All animal study protocols have been approved by the Office of Laboratory Animal Welfare, National Institute for Nutrition and Food Safety (Beijing, China). 2.1. Test substance L-Se-methylselenocysteine (SeMC), molecular weight 182.08, water-soluble white powder with purity >96%, was provided by Chuanqi Pharm Incoporation (Jiangxi, China).

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single gavage. General health observations and mortality were monitored on a daily base throughout 14 days after treatment. Median Lethal Dose (LD50) was calculated on the basis of animal death at different dose levels (Horn, 1956). 2.3. Genotoxicity study 2.3.1. Bacterial reverse mutation assay (Ames test) Four characterized histidine-dependent stains of Salmonella typhimurium (TA97, TA98, TA100, and TA102) were utilized for bacterial reverse mutation assay. S9 microsomal fraction of rat liver homogenate as the metabolic activation system was prepared according to previous method (Mortelmans and Zeiger, 2000). Bacteria in agar plate were treated with SeMC, solubilized in purified water, at concentrations of 0, 1.6, 8.0, 40, 200, 1000 lg/plate with or without S9 metabolic activation. The standard mutagens were used as positive controls in experiments, i.e. sodium azide (NaN3, 1.5 lg/plate) for TA100 without S9, 2-aminofluorene (2-AF, 10 lg/plate) for TA97, TA98, and TA100 with S9, 4-nitro-ophenylenediamine (4NOPD, 20 lg/plate) for TA97 and TA98 without S9, mitomycin (MMC, 2.5 lg/plate) for TA102 with S9, and 8-dihydroxyanthraquinone (DHAQ, 50 lg/plate) for TA102 without S9. After incubation for 48 h at 37 °C, the revertant colonies were counted manually. The experiment was repeated twice in the same condition. 2.3.2. Mouse bone marrow micronucleus assay Fifty BABL/c mice (six weeks old) were randomly divided into five groups, 10 mice per group and 5 mice for each sex. Animals were treated with SeMC at 1.15, 2.31 and 4.63 mg/kg BW by gavage, twice with 24 h interval. Cyclophosmide (40 mg/kg BW) was used as positive control while purified water as negative control. At 6 h after the second gavage, animals were euthanized and sternum aseptically removed. The contents of the spinal canal were squeezed out and diluted with calf serum, then smeared on the slides. After fixation with methanol and Giemsa staining, Red blood cells (RBC) and polychromatic erythrocytes (PCE) were observed under microscopy. The number of PCE was counted from 200 RBC in each animal and the ration of PCE/RBC was calculated. For each animal, 1000 PCE were examined for the incidence of micronucleated PCE. 2.3.3. Mouse sperm malformation assay Twenty-five male BABL/c mice (six weeks old) were randomly divided into five groups, 5 mice per group. Animals were treated with SeMC at 0, 1.15, 2.31 and 4.63 mg/kg BW, or cyclophosmide at 40 mg/kg BW by gavage, once a day for 5 days. Thirty days after the last gavage, animals were euthanized and both epididymides surgically removed. The epididymides were cut into pieces in saline then centrifuged at 1000 r/min for 7 min. The sperm suspension was applied on slide and dried in air. The slides were fixed with methanol and stained with 1.5% Eosin, and subsequently examined under microscopy. The spermatozoa with morphological abnormalities were counted from 1000 spermatozoa per animal for the calculation of malformation rates. 2.4. 90-day repeated dose study

2.2. Acute toxicity study The conventional method was used for the acute oral toxicity test of SeMC as described in Organization for Economic Co-operation and Development (OECD) guideline (OECD, 1987). Fifty BABL/c mice (six weeks old) were randomly divided into five groups, 10 mice per group and 5 mice for each sex. Base on the result of a range finding test, animals were treated with SeMC, solubilized in purified water, at 2.15, 4.64, 10.0, 21.5, 46.4 mg/kg BW by a

2.4.1. Study design Groups of 10 male and 10 female weaning Sprague–Dawley rats (six weeks old) were given SeMC by daily gavage at doses of 0.5, 0.7 and 0.9 mg/kg BW in a vehicle of purified water (5 ml/kg BW) for 90 days. The doses were designed according to the LD50 from acute oral toxicity study, and the highest dose was set as about 10% of LD50. Animals in the control group were given purified water for the same period. General clinical observations were recorded daily.

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Body weights and food consumption were measured weekly. Blood samples were obtained in the middle of the study (day 46) and at the end of the study (day 91) for examination of hematology and clinical chemistry. At the end of the study, all animals were weighed and euthanized for complete gross necropsy. Selected organs (liver, kidney, spleen, stomach, duodenum, heart, thymus, adrenals, testes, or ovary) were collected and weighed, then fixed in 10% formalin for histopathological examination. Organ-to-body weight ratios (relative organ weight) were calculated.

dyspnoea, diarrhea, tetanic spasms, and death. The mortality of animals at different doses was summarized in Table 1. Based on the analysis of dose-mortality relationship, the LD50 of SeMC was estimated as 12.6 mg/kg BW for female mice and 9.26 mg/kg BW for male mice. The values for acute toxicity are distributed in ‘‘category 2’’ (5–50 mg/kg BW) according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (UN, 2003), suggesting high health hazard of SeMC in the case of acute oral exposure.

2.4.2. Hematology and clinical biochemistry On days 46 and 91, rats were anesthetized with 3% sodium pentobarbital solution after 16–18 h fast and blood was collected from tail vein. The routine hematologic parameters such as red blood cell count (RBC), hemoglobin (HG), platelet count (PLT), white blood cells count (WBC), and leukocyte differential counts were measured with Coulter Diff Hematology Analyzer (Beckman Coulter Corporation) using the whole blood stabilized by the anticoagulant ethylenediaminetetraacetic acid (EDTA). Clinical chemistry was analyzed with automatic clinical analyzer (Hitachi 7080, Hitachi High-Technologies Corporation) to determine serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein, albumin, glucose, blood urea nitrogen, creatinine, cholesterol, and triglyceride.

3.2. Genotoxicity

2.4.3. Pathology All stored organs and tissues from each animal were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and subjected to microscopic examination. The incidences of pathologic lesions such as degeneration, necrosis, inflammation, cholangiolar changes, atrophy, hyperplasia, cellular infiltration were recorded as in each dosage group. 2.5. Statistical analysis Body weights, food consumption, clinical pathology, organ weights and Ames test data were expressed as mean ± standard deviation and evaluated using one-way analysis of variance (ANOVA). Dunnett’s test was used to analyze the significance of differences between control and treated groups. Incidence data including mouse micronucleated PCE and sperm malformation rates were analyzed by Fischer’s Exact Test. The significance level of P < 0.05 was used in all comparisons. 2.6. Benchmark dose–response modeling The US Environmental Protection Agency’s (EPA) benchmark dose software (BMDS) version 2.4.0 was used to estimate Benchmark Dose (BMD). For continuous data modeling, the ‘‘one standard deviation’’ was used as the default benchmark response (BMR) as recommended (EPA, 1995). BMDs and the 95% lower confidence values (BMDLs) were estimated by applying the dose–response models (i.e. Exponential, Power, Polynominal, Linear and Hill) available in the BMDS Software (EPA, 2011). To avoid ‘‘wavy’’ model responses on elevated biological changes in animals, adverse direction was restricted as ‘‘up’’ in Exponential, Linear, Power and Hill model, and the coefficients were restricted as ‘‘Non-negative’’ (>0) in Polynomial model. 3. Results 3.1. Acute toxicity After SeMC administration, clinical signs of poisoning were observed in mice, including dose-related decrease of body activity,

As shown in Table 2, SeMC treatment did not significantly increase the revertant colonies in plates of four strains TA97, TA98, TA100 and TA102 with or without S9 activation, respectively, when compared to negative controls. In contrast, the various mutagens induced dramatic increase of revertant colonies in relevant strains. Therefore, no mutagenic effect of SeMC was observed in Ames Test. Mouse bone marrow micronucleus assay showed that PCE/RBC ratios were not significantly suppressed by various doses of SeMC, indicating little cytotoxicity exerted by oral exposure (Table 3). As for MN incidence, CP treatment induced significant increase in animals of both sexes when compared with negative controls, however, no significant induction was observed in SeMC groups. The results indicated that SeMC was not mutagenic to somatic chromosomes in mouse. Cyclophosmide treatment significantly promoted sperm malformation in mouse; however, the incidence of abnormalities was not increased by SeMC at tested doses, suggesting SeMC had no adverse effect on the generation of spermatozoa in mouse (Table 4). Taken together, SeMC had no genotoxicity based on the multiple tests with in vitro and in vivo experiments. 3.3. Subchronic toxicity No mortality or treatment related adverse clinical appearances were found during the 90-day study. There were no statistically significant differences in animal body weights between the SeMC treatment groups and the control group (Fig. 1A). Similarly, no significant differences in food utilization (Fig. 1B) were observed between the SeMC groups and the control group of either sex. SeMC caused sporadic, statistically significant changes in hematology and clinical chemistry parameters (Tables 5 and 6). At the highest dose of SeMC (0.9 mg/kg BW), WBC was increased in female rats while HP was decreased in male rats when compared with the control group. However, the differences were not considered to be of toxicological significance since the changes were not dose-responsive and within the laboratory’s historical range of normal controls. No significant changes were observed in other parameters measured. More importantly, the liver weights and relative liver weights in female animals were significantly increased at 0.7 and 0.9 mg/kg BW (Table 7). In addition, the relative liver weights in male rats were also significantly increased by SeMC in a dose-dependent manner, with statistical significance at higher dosage levels (0.7 and 0.9 mg/kg BW). No obvious changes were found in other organs. No abnormalities were observed by gross necropsy in the animals which have undergone 90-day exposure to SeMC. Histopathological examination (Table 8) showed that sporadic cell necrosis in the liver was observed in limited number of animals receiving SeMC treatments, i.e. 2 rats at 0.9 mg/kg BW, 1 rat at each group of 0.5 and 0.7 mg/kg BW. However, the hepatic cell necrosis also observed in one animal in the control group. In addition, some animals were found suffering from slight myocardial necrosis in heart,

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H. Yang, X. Jia / Regulatory Toxicology and Pharmacology 70 (2014) 720–727 Table 1 Acute oral toxicity of SeMC in mice.

*

Animal gender

Dose (mg/kg BW)

N

Mortality

LD50 (mg/kg BW)

Toxicity category*

Female

2.15 4.64 10.0 21.5 46.4

5 5 5 5 5

0 0 2 4 5

12.6

2

Male

2.15 4.64 10.0 21.5 46.4

5 5 5 5 5

0 0 3 5 5

9.26

2

Toxicity was categorized according to the GHS classification.

Table 2 Effect of SeMC on bacterial reverse mutation (Ames test). Test agents

Dose (lg/ plate)

TA97

TA98

TA100

TA102

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

SeMC

0 1.6 8.0 40 200 1000

139.7 ± 15.5 149.7 ± 13.1 146.3 ± 12.7 139.3 ± 8.5 129.0 ± 18.0 134.0 ± 19.7

121.7 ± 9.0 121.7 ± 22.0 154.3 ± 22.5 131.0 ± 18.0 153.0 ± 21.9 132.0 ± 8.7

43.7 ± 6.7 51.3 ± 5.7 47.7 ± 11.8 50.3 ± 6.7 38.7 ± 6.4 42.3 ± 3.1

40.3 ± 11.0 53.3 ± 3.2 43.0 ± 3.0 51.0 ± 5.6 41.7 ± 7.1 47.7 ± 2.5

142.0 ± 19.9 121.3 ± 14.2 125.3 ± 17.6 131.3 ± 24.0 123.3 ± 13.1 122.3 ± 14.5

124.3 ± 4.2 117.0 ± 4.6 130.3 ± 22.0 140.0 ± 16.6 142.3 ± 12.7 132.0 ± 28.2

290.0 ± 20.1 193.7 ± 18.6 288.7 ± 22.1 284.7 ± 26.7 287.0 ± 26.6 289.0 ± 8.7

298.0 ± 19.5 270.3 ± 10.0 302.7 ± 17.9 276.3 ± 15.3 291.7 ± 12.7 278.7 ± 26.7

NaN3 2-AF 4NOPD MMC DHAQ

1.5 10.0 20.0 2.5 50.0

– – 1645.3 ± 92.6* –

– 1157.3 ± 139.1* – – –

– – 2181.3 ± 419.7* – –

– 2868.0 ± 223.0* – – –

1768.7 ± 136.2* – – – –

– 1698.7 ± 276.2* – – –

– – – 2483.3 ± 316.3* –

– – – – 874.3 ± 85.0*

NaN3, Sodium azide; 2-AF, 2-aminofluorene; 4NOPD, 4-nitro-o-phenylenediamine; MMC, mitomycin; DHAQ, 8-dihydroxyanthraquinone. Data were analyzed with ANONA followed by Dunnett’s test for multiple comparison, all values from triplicate plates are expressed as mean ± S.D. * P < 0.05, significantly different as compared with the controls.

Table 3 Effect of SeMC on micronucleus induction in mouse bone marrow. Animal gender

Female

Male

Group (mg/kg BW)

SeMC 0 SeMC 1.16 SeMC 2.32 SeMC 4.63 CP 40.0 SeMC 0 SeMC 1.16 SeMC 2.32 SeMC 4.63 CP 40.0

N

5 5 5 5 5 5 5 5 5 5

PCE analysis

MN analysis

RBC

PCE

PCE/RBC (%)

PCE number

MN number

MN/PCE (‰)

200 200 200 200 200 200 200 200 200 200

109.4 ± 6.0 105.4 ± 4.5 108.4 ± 3.2 107.2 ± 5.1 99.6 ± 8.3 110.0 ± 8.5 107.8 ± 5.6 110.2 ± 3.4 107.0 ± 5.8 97.2 ± 12.6

54.7 52.7 54.2 53.6 49.8 55.0 53.9 55.1 53.5 48.6

1000 1000 1000 1000 1000 1000 1000 1000 1000 1000

0.8 ± 0.8 1.0 ± 1.0 0.8 ± 0.8 1.0 ± 1.0 24.0 ± 8.1* 0.8 ± 0.8 0.8 ± 0.8 0.6 ± 0.6 0.8 ± 0.8 21.8 ± 5.9*

0.8 1.0 0.8 1.0 24.0* 0.8 0.8 0.6 0.8 21.8*

CP, cyclophosmide; PCE, polychromatic crythrocyte; RBC, red blood cells; MN, micronucleus. Five animals were treated in each group; MN incidence (MN/PCE) was analyzed with Fisher’s exact test. * P < 0.05, significantly different as compared with the controls.

i.e. 3 rats at 0.9 mg/kg BW, 4 rats at 0.7 mg/kg BW, 2 rats at 0.5 mg/ kg BW, and 1 rat in the control group. However, there was no statistically significant difference between the control and treatment groups. No pathological changes of biological significance were observed in all other tissues. 3.4. BMD calculation Data on relative liver weight was evaluated for BMD calculations since the liver appeared to be the most sensitive target organ for SeMC toxicity. The calculations of BMD or BMDL values are summarized in Table 9. According to the tests for goodness

of fit, Exponential 5 and Hill model were acceptable to describe the data for both male and female rats (Supplemental Tables 1 and 2). Then, as recommended by US EPA (EPA, 1995, 2011), Akaike’s Information Criterion (AIC) was used for model comparison and Hill model was identified to be used for BMDL calculation. Finally, the BMDLs were determined as 0.47 mg/kg BW (Fig. 2A) and 0.34 mg/kg BW (Fig. 2B) for female and male animals, respectively. To obtain an heath protection for whole population, BMDL = 0.34 mg/kg could be conservatively used as the point of departure to estimate the health-based guidance values such as a Reference Dose (RfD) or Acceptable Daily Intake (ADI).

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Table 4 Effect of SeMC on sperm malformation in mouse. Group (mg/kg BW)

N

Number of spermatozoa

Number of malformation

Malformation incidence (%)

SeMC 0 SeMC 1.16 SeMC 2.32 SeMC 4.63 CP 40.0

5 5 5 5 5

1000 1000 1000 1000 1000

21.4 ± 3.0 20.2 ± 3.1 21.4 ± 3.0 19.2 ± 4.1 52.2 ± 7.9

2.14 2.02 2.14 1.92 5.22*

Five animals were treated in each group; Sperm malformation rate was analyzed with Fisher’s exact test. * P < 0.05, significantly different as compared with the controls.

Fig. 1. General observation on animals in the subchronic oral toxicity study of SeMC. (A) Body weight changes. (B) Comparison on food utilization rates.

Table 5 Effect of SeMC on hematology in rats. Animal gender

Dose (mg/kg BW)

N

Blood routine parameters 9

Leukocyte differential count (%) 12

12

WBC (10 /L)

RBC (10 /L)

PLT (10 /L)

HB (g/L)

Lymphocyte

Neutrophile

Other

Female

0.0 0.5 0.7 0.9

10 10 10 10

7.90 ± 2.33 9.34 ± 2.59 10.78 ± 4.4 12.99 ± 5.0*

6.94 ± 0.27 6.86 ± 0.45 6.87 ± 0.53 6.72 ± 0.41

892.3 ± 116.5 975.7 ± 130.0 856.0 ± 185.2 874.7 ± 257.0

155.0 ± 4.3 152.1 ± 5.6 151.4 ± 8.6 147.3 ± 4.6

72.4 ± 4.9 77.1 ± 4.7 77.2 ± 3.5 74.6 ± 5.0

19.6 ± 5.1 15.7 ± 4.7 15.5 ± 4.3 18.8 ± 5.3

8.0 ± 2.3 7.2 ± 1.0 7.3 ± 2.0 6.6 ± 1.5

Male

0.0 0.5 0.7 0.9

10 10 10 10

11.03 ± 4.34 13.79 ± 4.62 13.69 ± 4.97 11.97 ± 3.23

7.29 ± 0.30 7.32 ± 0.23 7.45 ± 0.27 7.12 ± 0.30

911.7 ± 100.8 886.1 ± 90.8 844.8 ± 137.1 842.3 ± 275.3

158.4 ± 5.7 155.5 ± 5.0 156.3 ± 5.0 149.4 ± 3.7*

73.9 ± 4.6 69.9 ± 4.3 72.7 ± 4.2 71.4 ± 4.1

18.4 ± 3.5 22.5 ± 3.8 20.2 ± 4.6 21.2 ± 4.1

7.7 ± 2.9 7.6 ± 2.1 7.1 ± 1.2 7.4 ± 1.6

WBC, white blood cell (leukocyte) count; RBC, red blood cell (erythrocyte) count; PLT, platelet count; HB, hemoglobin. Data were analyzed with ANONA followed by Dunnett’s test for multiple comparison, all values are expressed as mean ± S.D. of each group. * P < 0.05, significantly different as compared with the controls.

4. Discussion Our results demonstrated that SeMC, an organic selenium compound with promising anticancer activity, is of high potent of health hazard to human under acute oral exposure, but not likely to induce genotoxicity. The subchronic toxicity study indicated significantly less systemic toxicity of SeMC at supernutritional selenium levels (over 10 times the requirement) as compared to other selenium compounds such as selenate. Importantly, the experiment identified liver as the primary target organ of SeMC toxicity in rats on the basis of elevated relative liver weight after 90-day exposure. In addition, the BMDL of 0.34 mg/kg BW/day was estimated for the risk assessment of its use as food supplement or pharmaceutical agent. Selenium toxicity is largely dependent on its chemical form. An comparative study concerning the oral toxicity of various selenium forms showed that the least toxic selenium compound was insoluble elemental selenium with an LD50 of 6700 mg/kg BW while the

most toxic compound was the highly soluble sodium selenite with an LD50 of 7 mg/kg BW, indicating that the broad variations in LD50 values was associated with the aqueous solubility (Cummins and Kimura, 1971). On the other hand, the minimum lethal dose of soluble selenium as sodium selenite or selenate in rabbits, rats, and cats was within 1.5–3.0 mg/kg BW, regardless of whether the compounds were administered orally, subcutaneously, intraperitoneally, or intravenously (WHO/IPCS, 1987). This lack of effect of the mode of administration probably reflects the rapid and complete absorption of soluble selenium compounds, either from the site of injection or from the gastrointestinal tract. The LD50 of SeMet in rats given an intraperitoneal injection was determined as 4.25 mg Se/kg BW and is comparable to that of selenite or selenate (Schrauzer, 2000). In our study, SeMC was also highly soluble in water and the oral LD50 obtained from mouse was 9.26– 12.60 mg/kg BW, implicating SeMC is slightly less toxic than sodium selenite which had been previously tested in male mouse with an oral LD50 of 7.08–7.75 mg/kg BW (WHO/IPCS, 1987).

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H. Yang, X. Jia / Regulatory Toxicology and Pharmacology 70 (2014) 720–727 Table 6 Effect of SeMC on clinical biochemistry in rats. Animal gender

Dose (mg/kg BW)

N

ALT (U/L)

AST (U/L)

Female

0.0 0.5 0.7 0.9

10 10 10 10

37.3 ± 6.5 38.5 ± 8.2 31.9 ± 7.4 39.3 ± 10.8

144.5 ± 22.4 158.9 ± 24.2 138.6 ± 24.2 154.8 ± 19.4

Male

0.0 0.5 0.7 0.9

10 10 10 10

33.4 ± 4.3 40.7 ± 17.4 40.4 ± 5.9 40.7 ± 8.5

140.7 ± 26.4 165.4 ± 25.8 159.7 ± 31.2 157.9 ± 18.6

BUN (mmol/L)

SCr (lmol/L)

CHO (mmol/L)

TG (mmol/L)

GLU (mmol/L)

TP (g/L)

Alb (g/L)

56.7 ± 13.4 67.4 ± 16.2 79.0 ± 21.6 72.6 ± 20.3

5.72 ± 0.71 6.34 ± 1.15 6.41 ± 0.85 5.11 ± 0.92

65.7 ± 5.0 69.8 ± 6.8 65.3 ± 6.3 64.4 ± 6.7

2.04 ± 0.33 2.12 ± 0.38 2.29 ± 0.24 1.78 ± 0.39

0.58 ± 0.22 0.57 ± 0.20 0.75 ± 0.25 0.72 ± 0.27

5.68 ± 0.54 5.68 ± 0.53 6.00 ± 0.81 5.40 ± 0.86

77.1 ± 4.5 78.3 ± 5.3 78.0 ± 4.2 74.6 ± 1.9

39.3 ± 2.3 40.3 ± 2.6 39.4 ± 1.5 37.6 ± 2.5

86.4 ± 27.1 98.4 ± 13.9 92.6 ± 17.6 106.7 ± 14.5

6.57 ± 1.37 5.46 ± 0.70 6.16 ± 0.89 6.04 ± 1.05

65.8 ± 6.2 63.6 ± 3.4 65.0 ± 6.1 59.1 ± 10.6

1.67 ± 0.44 1.43 ± 0.24 1.44 ± 0.22 1.38 ± 0.18

0.79 ± 0.32 0.68 ± 0.24 0.64 ± 0.14 0.75 ± 0.21

5.65 ± 0.51 5.57 ± 0.45 5.38 ± 0.88 5.41 ± 0.27

75.7 ± 3.2 74.6 ± 4.5 72.3 ± 4.6 72.6 ± 7.0

36.1 ± 2.0 36.5 ± 2.1 36.9 ± 2.0 36.6 ± 2.1

ALP (U/L)

ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; BUN, urea nitrogen; SCr, serum creatinine; CHO, cholesterol; TG, triglycerides; GLU, Glucose; TP, total protein; Alb, albumin. Data were analyzed with ANONA followed by Dunnett’s test for multiple comparison, all values are expressed as mean ± S.D. of each group.

Table 7 Effect of SeMC on relative organ weight in rats. Animal gender

Dose (mg/kg BW)

N

Body weight (g)

Female

0.0 0.5 0.7 0.9

10 10 10 10

274.7 ± 22.3 259.6 ± 28.4 257.6 ± 24.2 267.4 ± 29.9

Male

0.0 0.5 0.7 0.9

10 10 10 10

469.2 ± 44.3 452.5 ± 37.9 448.2 ± 47.3 448.7 ± 42.9

Female

0.0 0.5 0.7 0.9

10 10 10 10

Male

0.0 0.5 0.7 0.9

10 10 10 10

Liver Weight (g)

Kidney

Spleen

%

Weight (g)

%

Weight (g)

%

2.64 ± 0.20 2.84 ± 0.38 3.56 ± 0.25* 3.63 ± 0.38*

2.08 ± 0.24 2.12 ± 0.27 2.12 ± 0.25 2.16 ± 0.15

0.76 ± 0.07 0.83 ± 0.15 0.82 ± 0.08 0.82 ± 0.08

0.60 ± 0.07 0.57 ± 0.09 0.61 ± 0.12 0.59 ± 0.21

0.22 ± 0.02 0.22 ± 0.04 0.23 ± 0.03 0.22 ± 0.07

12.47 ± 1.18 12.65 ± 1.43 13.45 ± 1.93 13.99 ± 1.88

2.66 ± 0.13 2.80 ± 0.19 2.99 ± 0.17* 3.12 ± 0.24*

3.42 ± 0.36 3.33 ± 0.32 3.30 ± 0.32 3.26 ± 0.49

0.73 ± 0.07 0.74 ± 0.10 0.74 ± 0.05 0.73 ± 0.09

0.85 ± 0.09 0.85 ± 0.16 0.86 ± 0.17 0.78 ± 0.16

0.18 ± 0.02 0.19 ± 0.04 0.19 ± 0.03 0.17 ± 0.03

274.7 ± 22.3 259.6 ± 28.4 257.6 ± 24.2 267.4 ± 29.9

0.43 ± 0.10 0.36 ± 0.10 0.41 ± 0.09 0.35 ± 0.08

0.16 ± 0.04 0.14 ± 0.04 0.16 ± 0.02 0.13 ± 0.03

1.09 ± 0.08 1.00 ± 0.12 1.07 ± 0.07 1.09 ± 0.15

0.40 ± 0.03 0.39 ± 0.06 0.42 ± 0.04 0.41 ± 0.05

– – – –

– – – –

469.2 ± 44.3 452.5 ± 37.9 448.2 ± 47.3 448.7 ± 42.9

0.57 ± 0.09 0.53 ± 0.11 0.49 ± 0.10 0.50 ± 0.10

0.12 ± 0.02 0.12 ± 0.03 0.11 ± 0.02 0.10 ± 0.04

1.67 ± 0.19 1.58 ± 0.14 1.53 ± 0.14 1.54 ± 0.18

0.36 ± 0.03 0.35 ± 0.03 0.34 ± 0.03 0.34 ± 0.04

3.65 ± 0.27 3.96 ± 0.88 3.52 ± 0.39 3.52 ± 0.25

0.78 ± 0.07 0.88 ± 0.17 0.79 ± 0.11 0.79 ± 0.06

7.26 ± 0.78 7.33 ± 0.86 9.16 ± 0.97* 9.70 ± 1.46*

Data were analyzed with ANONA followed by Dunnett’s test for multiple comparison, all values are expressed as mean ± S.D. of each group; * P < 0.05, significantly different as compared with the controls.

Table 8 Histopathology examination on rats after SeMC treatment. Microscopic lesions

Liver

Kidney

Heart

Dose (mg/kg BW)

Degeneration Necrosis Inflammation Cholangiolar changes Glomerular atrophy Inflammatory exudate Papillary necrosis Interstitial cell hyperplasia Myocardial necrosis Degeneration Cellular infiltration

Other tissues Spleen (spleen, stomach, duodenum, thymus, adrenals, testes, ovary)

0

0.5

0.7

0.9

0 1/20 0 0 0 0 0 0 1/20 0 0 0

0 1/20 0 0 0 0 0 0 2/20 0 0 0

0 1/20 0 0 0 0 0 0 4/20 0 0 0

0 2/20 0 0 0 0 0 0 3/20 0 0 0

Tissues from twenty animals were examined in each group.

Genotoxicity of selenium has been tested in a great variety of in vitro assays, results of which emphasized the importance of the chemical form and also dose in the effects. Letavayová et al. compared the genotoxicity of selenium compounds including sodium selenite, selenomethionine, and SeMC in Saccharomyces cerevisiae (yeast) (Letavayova et al., 2008). The results showed that only sodium selenite manifested a significant toxic effect which

was likely associated with the oxidative damage to DNA. Selenium-enriched yeast with the selenomethionine content greater than 98% was proved nongenotoxic by a battery of assays(Griffiths et al., 2006). But some organoselenium compounds were reported genotoxic to human leukocytes cells in vitro when tested in relatively high concentrations (100 lM), and this effect was suggested to be linked to the pro-oxidant activity (Santos

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Table 9 BMDs and BMDLs based on the relative liver weight from 90-day repeated dose study. Test models

Female

Male *

Exponential2 Exponential3 Exponential4 Exponential5 Hill Linear Polynomial Power

P for Fit

BMD

BMDL

P for Fit*

BMD

BMDL

0.003 0.002 0.000 NA# NA# 0.002 0.002 0.002

0.32 0.44 0.30 0.51 0.52 0.30 0.46 0.46

0.26 0.28 0.23 0.45 0.47 0.23 0.28 0.28

0.305 0.464 0.096 NA# NA# 0.251 0.462 0.480

0.37 0.52 0.36 0.52 0.54 0.36 0.52 0.52

0.28 0.32 0.27 0.32 0.34 0.27 0.32 0.32

* P > 0.1 implies that the model seems to adequately describe the data according to the US EPA BMDS Software. # Not applicable: degrees of freedom for test are less than or equal to 0, the test for model fit is not valid.

et al., 2009). Results from other studies using chromosome aberrations test, sister chromatid exchanges (SCE) test and micronucleus (MN) test are currently controversial (Valdiglesias et al., 2010), with the genotoxicity of SeMC poorly characterized. In this study, SeMC showed no mutagenic activity in Ames test in vitro. In vivo, SeMC did not induce mouse bone marrow micronucleus or sperm malformation even at high dosage levels (1/2 LD50). Collectively, these results provided strong evidence indicating the low risk of SeMC genotoxicity under oral exposure. Animal studies have demonstrated that the liver is the major target organ of selenium toxicity (WHO/IPCS, 1987). A subacute toxicity study found that SeMC induced dose-related hepatomegaly and hepatocellular degeneration in rats and dogs after 28-day treatment (Johnson et al., 2008). In agreement, our 90-day subchronic toxicity study demonstrated that the relative liver weight was dose-dependently increased by SeMC in both male and female rats. In addition, myocardial necrosis observed in the pathological examinations implied potential toxicity of SeMC targeting the heart, though there was no statistically significant difference between the control and treatment groups. Long-term intake of selenium at supernutritional levels is the major route to cause the risk of adverse health effects such as selenosis. The current requirement of selenium in AIN-93 diets for laboratory rodents is 0.15 ppm and the recommended form of added dietary selenium is selenate (Reeves et al., 1993). Previous studies have shown that chronic toxicity of dietary selenite begins at 3–5 ppm (Jia et al., 2005). The survival age of rats fed 16 ppm sodium selenate (represents an intake of selenium about 0.4 mg/ kg BW per day) was reported only 96 days and only 4 ppm was needed to cause histopathological lesion in the liver (WHO/IPCS, 1987). Due to the significant toxicity, US FDA required the upper

limit of selenium in complete feed for chickens, swine, turkeys, sheep, cattle, and ducks not to exceed 0.3 ppm (FDA, 1997). At present, limited data is available to assess the chronic oral toxicity of SeMC. In this study, no evidence of health hazard was identified during clinical observations of rats exposed to any levels of SeMC. In addition, animal growth which was recognized as the best indicator of chronic selenium toxicity (NAS/NRC, 1976) was not significantly affected by daily oral exposure at levels up to 0.9 mg/kg BW (equal with an intake of selenium about 0.4 mg/kg BW), suggesting much lower system toxicity of SeMC as compared to selenate or selenite. On the other hand, we previously determined the No Observed Adverse Effect Level (NOAEL) for selenite and highselenium protein at 0.14 mg Se/kg BW/day based on a 90-day feeding study (Jia et al., 2005). In this study, the NOAEL for SeMC can be estimated at 0.5 mg/kg BW/day (0.22 mg Se/kg BW/day), also suggesting slightly lower toxicity in comparison with selenite and high-selenium protein. SeMC differs from inorganic selenocompounds or other organoselenium largely in its methylated products (CH3SeH), which may contributes to the relatively lower toxicity as well as the anticancer activity (Hoefig et al., 2011; Ip et al., 1991). For the recognized limitations in the conventional NOAEL or LOAEL approach, BMD approach is now increasingly utilized for the toxicological dose–response assessment in health risk evaluation (Muri et al., 2009). In this study, through BMD modeling with the data on relative liver weight, the BMDL for SeMC was determined as 0.34 mg/kg BW/day (about 30% lower than the NOAEL value) to ensure adequate safety margin. This level is equivalent with the selenium content of about 2 ppm in animal feed, which is still more than 10 times the requirement of 0.15 ppm, suggesting the superior safety of SeMC as a nutritional selenium supplement. However, this level is quiet close to the effective dose of SeMC (2 ppm selenium in diet) that has been usually used in animal studies for cancer prevention. Therefore, more detailed studies are needed to revalidate the efficacy of SeMC in health protection against cancers while avoiding chronic toxicity. Although the BMDL was based on subchronic data in this study, it is not likely that extending of exposure to chronic duration at the estimated BMDL will generate significant extra-toxicity according to the metabolic characteristic of SeMC and the function of animal body maintaining selenium homeostasis (Hoefig et al., 2011; Ip et al., 1991). Therefore, a default uncertainty factor of 100 was applied for the result extrapolation from rats to human, and the ADI for SeMC can be estimated as 3.4 lg/kg BW/day. To avoid the potential chronic toxicity, it limits an intake of SeMC not to exceed 240 lg per day by an adult with 70 kg body weight. This level equals a supplemental dose of selenium about 100 lg per day, which would result in the total daily intake of selenium up

Fig. 2. Benchmark dose modeling for the relative liver weight of rats in the subchronic oral toxicity study of SeMC. (A) Hill model fitting curve for female data. (B) Hill model fitting curve for male data.

H. Yang, X. Jia / Regulatory Toxicology and Pharmacology 70 (2014) 720–727

to 250 lg for an average adult. No matter of the correlation between SeMC toxicity and selenium content, this is a safe amount since it is significantly below the RfD for selenium, which was set by the EPA at 350 lg per day (Patterson and Levander, 1997). In summary, SeMC revealed no genotoxicity and minor subchronic oral toxicity at doses far beyond the nutritional selenium level. On the basis of dose–response characterization via BMD approach, the estimation of ADI at 3.4 lg/kg BW/day for SeMC dietary intake was derived and provided further safety warrant for its use as nutritional selenium supplement. But the health risk should be carefully assessed when the use of SeMC is aiming to achieve cancer chemoprevention. Thus, more detailed toxicological studies are needed to evaluate the efficacy and safety of SeMC as a promising anticancer agent. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.yrtph.2014.10. 014. References Alfthan, G., Eurola, M., Ekholm, P., Venalainen, E.R., Root, T., Korkalainen, K., Hartikainen, H., Salminen, P., Hietaniemi, V., Aspila, P., Aro, A., 2014. Effects of nationwide addition of selenium to fertilizers on foods, and animal and human health in Finland: From deficiency to optimal selenium status of the population. J. Trace Elem. Med. Biol. Bratter, P., Negretti de Bratter, V.E., 1996. Influence of high dietary selenium intake on the thyroid hormone level in human serum. J. Trace Elem. Med Biol. 10, 163– 166. Brown, K.M., Arthur, J.R., 2001. Selenium, selenoproteins and human health: a review. Public Health Nutr. 4, 593–599. Cao, S., Durrani, F.A., Toth, K., Rustum, Y.M., 2014. Se-methylselenocysteine offers selective protection against toxicity and potentiates the antitumour activity of anticancer drugs in preclinical animal models. Br. J. Cancer 110, 1733–1743. Chen, J., 2012. An original discovery: selenium deficiency and Keshan disease (an endemic heart disease). Asia Pac J Clin Nutr. 21, 320–326. Cummins, L.M., Kimura, E.T., 1971. Safety evaluation of selenium sulfide antidandruff shampoos. Toxicol. Appl. Pharmacol. 20, 89–96. El-Bayoumy, K., Sinha, R., 2004. Mechanisms of mammary cancer chemoprevention by organoselenium compounds. Mutat. Res. 551, 181–197. EPA, 1995. The Use of the Benchmark Dose Approach in Health Risk Assessment. United States Environmental Protection Agency, Washington, DC. EPA, 2011. User manual for BMDS 2.2. United States Environmental Protection Agency, Washington, DC. FDA, 1997. Food additives permitted in feed and drinking water of animals; selenium. Fed. Reg. 62, 44892–44893. FDA, 2000. Toxicological Principles for the Safety Assessment of Food Ingredients Redbook 2000. College Park, MD. FNB, 2000. Selenium. In: Food and Nutrition Board (Ed.) Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and Carotenoids National Academy Press, Washington DC, pp. 284–324. Freeman, J.L., Marcus, M.A., Fakra, S.C., Devonshire, J., McGrath, S.P., Quinn, C.F., Pilon-Smits, E.A., 2012. Selenium hyperaccumulator plants Stanleya pinnata and Astragalus bisulcatus are colonized by Se-resistant, Se-excluding wasp and beetle seed herbivores. PLoS ONE 7, e50516. Griffiths, J.C., Matulka, R.A., Power, R., 2006. Genotoxicity studies on Sel-Plex, a standardized, registered high-selenium yeast. Int. J. Toxicol. 25, 477–485. Hoefig, C.S., Renko, K., Kohrle, J., Birringer, M., Schomburg, L., 2011. Comparison of different selenocompounds with respect to nutritional value vs. toxicity using liver cells in culture. J. Nutr. Biochem. 22, 945–955. Horn, H.J., 1956. Simplified LD50 (or ED50) calculations. Biometrics 12, 311–322. Ip, C., Hayes, C., Budnick, R.M., Ganther, H.E., 1991. Chemical form of selenium, critical metabolites, and cancer prevention. Cancer Res. 51, 595–600. Ip, C., Thompson, H.J., Zhu, Z., Ganther, H.E., 2000. In vitro and in vivo studies of methylseleninic acid: evidence that a monomethylated selenium metabolite is critical for cancer chemoprevention. Cancer Res. 60, 2882–2886. Ip, C., Dong, Y., Ganther, H.E., 2002. New concepts in selenium chemoprevention. Cancer Metastasis Rev. 21, 281–289. Jia, X., Li, N., Chen, J., 2005. A subchronic toxicity study of elemental Nano-Se in Sprague-Dawley rats. Life Sci. 76, 1989–2003.

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