Food and Chemical Toxicology 75 (2015) 146–150
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
A subchronic oral toxicity study on pyrroloquinoline quinone (PQQ) disodium salt in rats Chunlai Liang, Xin Zhang, Wei Wang, Yan Song, 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
I N F O
Article history: Received 4 June 2014 Accepted 7 November 2014 Available online 15 November 2014 Keywords: Pyrroloquinoline quinone disodium salt Subchronic toxicity Rats NOAEL
A B S T R A C T
A subchronic oral toxicity study on pyrroloquinoline quinone (PQQ) disodium salt was performed in rats. Sprague-Dawley rats were randomly divided into four groups (10 rats/sex/group) and administered with PQQ disodium salt at doses of 0 (control), 100, 200 and 400 mg/kg bw/day by gavage for 13 weeks. Daily clinical observations and weekly measurement of body weights and food consumption were conducted. Blood samples were obtained on day 46 and day 91 for measurement of hematology and serum biochemical parameters. Animals were euthanized for necropsy, selected organs were weighted and recorded. Histological examination was performed on all tissues from animals in the control and PQQ disodium salt treatment groups. No mortality or toxicologically significant changes in clinical signs, body weight, food consumption, necropsy findings or organ weights was observed. Differences between treated and control groups in some hematological and serum biochemical examinations and histopathological examination were not considered treatment-related. The no-observed-adverse-effect-level (NOAEL) of PQQ disodium salt in rats was considered to be 400 mg/kg bw/day for both sexes, the highest dose tested. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction In 1979, pyrroloquinoline quinone (PQQ) was identified as a novel coenzyme in several bacterial dehydrogenases (Salisbury et al., 1979). Since then, it has been detected in various foods and in tissues and body fluids of humans and rats (Kumazawa et al., 1992, 1995; Noji et al., 2007). PQQ can be synthesized by some microorganisms, the major source of PQQ in human is believed to be microbial sources from the diet (Kumazawa et al., 1995; Misra et al., 2012). PQQ plays an important nutritional role in both bacteria and higher organisms and is involved in numerous physiological and biochemical processes (Misra et al., 2012; Rucker et al., 2009). It has been reported to have benefits in rodents related to improvement of neonatal growth, reproductive performance and immune function, as well as cardioprotection and neuroprotection (Steinberg et al., 1994, 2003; Tao et al., 2007; Zhang et al., 2006).
Abbreviations: ALB, albumin; ALT, alanine aminotransferase; ALP, alkaline phosphatase; ANOVA, analysis of variance; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CHO, cholesterol; CRE, creatinine; FDA, Food and Drug Administration; GLU, glucose; GR, granulocyte; HGB, hemoglobin; LYM, lymphocyte; MO, monocyte; NOAEL, no-observed-adverse-effect-level; PLT, platelet count; PQQ, pyrroloquinoline quinone; RBC, red blood cell count; SD rat, Sprague-Dawley rat; TG, triglyceride; TP, total protein; WBC, white blood cell count. * Corresponding author. Key Laboratory of Food Safety Risk Assessment of Ministry of Health, National Center for Food Safety Risk Assessment, Beijing 100021, China. Tel./fax: +86-10-67770977. E-mail address: [email protected] (X. Jia). http://dx.doi.org/10.1016/j.fct.2014.11.005 0278-6915/© 2014 Elsevier Ltd. All rights reserved.
In 2003, two scientists identified a PQQ-dependent dehydrogenase enzyme in mice and found that PQQ acted as a mammalian redox cofactor in lysine metabolism. Thus they concluded PQQ to be a newcomer to the B group of vitamins (Kasahara and Kato, 2003). However, the statement was subsequently doubted by other scientists who claimed that sufficient evidence was not available to conclude that PQQ performed an essential vitamin function in mammals (Felton and Anthony, 2005; Rucker et al., 2005). Nevertheless, PQQ did appear to have growth promoting properties in rodents and there are several patents on PQQ applications in different countries including the United States of America, Japan and China focusing on its commercial potential as human food and/or supplement ingredient (Gu et al., 2012; Sumi et al., 2012; Tsuji et al., 1998; Zhou et al., 2010). For any new dietary ingredient intended to be added into foods or used as a dietary supplement, it is necessary and essential to conduct a safety assessment, especially a toxicological evaluation. However, there are only a few reports on the toxicity evaluation of PQQ. One study evaluated the potential genotoxicity of PQQ disodium salt using a battery of genotoxicity tests (Ames test, in vitro chromosomal aberration test in Chinese hamster lung cells, in vitro chromosomal aberration test in human peripheral blood lymphocytes and in vivo micronucleus assay in mice) and concluded that PQQ had no genotoxic activity (Nakano et al., 2013). In another study, functional and morphologic changes of kidneys were observed when PQQ was intraperitoneally injected into rats at a dose of 11.5 mg/ kg (Watanabe et al., 1989). Subchronic toxicity studies can provide more information on the possible health hazards of test
C. Liang et al./Food and Chemical Toxicology 75 (2015) 146–150
substances from repeated exposure over a prolonged period of time; therefore, a 13-week subchronic oral toxicity test on PQQ disodium salt was conducted in rats in this study. 2. Materials and methods 2.1. Test substance PQQ disodium salt, molecular weight 374.17, water-soluble reddish brown crystalline powder with mild hydroscopic property, purity >98%, was provided by Shanghai Med Co., Ltd., China.
2.2. Animals Weanling Sprague-Dawley (SD) rats, specific pathogen-free grade, were purchased from Vital River Laboratory Animal Technology Co, Ltd (Beijing, China). Body weights of rats at receipt were 60–80 g. All animals were examined for clinical signs of ill health on receipt and observed within 5 days of arrival. Rats were housed in an environmental controlled room with the temperature at 23 ± 2 °C and the relative humidity within the range of 40–70%. Air was changed 10–15 times per hour. Light was set for a 12 hr light/dark cycle. Rats were individually housed in suspended stainless steel, open-mesh cages and allowed free access to pellet feed and tap water during the experiments.
2.3. Experimental design Eighty healthy SD rats were randomly divided into four groups, 10 animals/sex/ group. One group administered with water by gavage served as the control. An acute oral toxicity of PQQ disodium salt was previously conducted by Horn’s method as described in the Chinese Toxicology Assessment Procedures and Methods for Food Safety (Chinese Standard GB15193.3-2003) in our lab, the dose selection for this study was based on the result of the acute oral toxicity (the LD50 of females and males was 5.01 and 3.69 g/kg, respectively), 400 mg/kg bw/day (around 10% of LD50) was selected as the high dose level, and 200 mg/kg bw/day and 100 mg/kg bw/day were chosen as the other two lower levels. PQQ disodium salt was administered to rats by gavage once daily for 13 weeks. Clinical observations were recorded daily. Body weights were measured weekly and the gavage volume (1 ml/100 g body weight) was adjusted accordingly. Blood samples were obtained in the middle of the study (day 46) and at the end of the study for measurement of hematology and serum biochemistry parameters. At the end of the study, all animals were euthanized for necropsy. Selected organs were weighted and recorded. Histological examination was performed on all tissues from animals in the control group and PQQ disodium salt treatment groups. The study was 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 (US Food and Drug Administration (FDA), 2007). The protocol has been approved by the Office of Laboratory Animal Welfare, National Institute for Nutrition and Food Safety.
2.4. Clinical observations
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2.7. Pathology All rats were humanely sacrificed at the end of the test, and a complete necropsy was performed including an examination of the external features of the carcass, external body orifices, the abdominal, thoracic, and cranial cavities, organs and tissues. Organ weights were obtained for the heart, kidneys, liver, spleen, testes and thymus. Paired organs were weighed together. Organ-to-body weight ratios (relative organ weights) were also calculated. In addition to the above-mentioned organs, the following tissues (when present) were sampled and fixed in 10% neutral-buffered formalin: cecum, colon, duodenum, esophagus, femur with bone marrow, ileum, jejunum, lacrimal gland, lung, lymph node, mammary gland, nasal turbinates, pancreas, pituitary gland, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin, spinal cord, sternum with bone marrow, trachea, urinary bladder, vagina, ovary and adrenals. All stored organs and tissues from each animal in the control group and PQQ disodium salt treatment groups were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and subjected to microscopic examination. Macroscopic lesions observed at necropsy were also examined from each animal in all groups. 2.8. Statistical analysis Data were presented as mean ± SD. The SPSS Statistical System (SPSS for Windows 18.0, Chicago, USA) was used to analyze body weights, food consumption, hematology and serum biochemistry parameters, and organ weights, followed by testing for variance homogeneity. Homogeneous data were analyzed using the analysis of variance (ANOVA) and the significance of differences between control and treated groups were analyzed using Dunnett’s multiple comparisons. P < 0.05 was considered statistically significant.
3. Results 3.1. Mortality and clinical signs No mortality or treatment related adverse clinical reactions were found during the study. 3.2. Body weight and food consumption There were no statistically significant differences in body weights between the treatment groups and the control group in each week (Fig. 1). No significant differences were observed between the treatment groups and the control group for weekly food consumption of females and males (Fig. 2). 3.3. Hematology and serum biochemistry There were some sporadic, statistically significant changes in some hematology and serum biochemical parameters (Tables 1 and
Each animal was observed twice daily for abnormalities, physical appearance and mortality throughout the study. Observations included, but were not limited to, changes in skin, fur, eyes, appearance, salivary gland secretions, oral mucosa, fecal characteristics, respiration and behavior.
2.5. Body weight and food consumption The body weights of rats were measured pre-test, weekly thereafter and at sacrifice after fasting. Food consumption was measured once a week during the experiment period.
2.6. Hematology and serum biochemistry On day 46 and day 91, rats were anesthetized with 3% sodium pentobarbital solution and blood samples were collected from the tail vein. Blood samples for hematology were collected into tubes containing ethylenediaminetetraacetic acid anticoagulant. A COULTER Ac.T diff2 Hematology Analyzer (Beckman Coulter Corporation) was employed to measure the following parameters: red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), white blood cell count (WBC) and WBC differential count of lymphocyte (LYM), granulocyte (GR) and monocyte (MO). Blood samples for serum biochemistry were collected into tubes containing no anticoagulant and centrifuged to obtain serum. An automatic analyzer (Hitachi 7080, Hitachi High-Technologies Corporation) was used to analyze the following parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), glucose (GLU), blood urea nitrogen (BUN), creatinine (CRE), cholesterol (CHO) and triglyceride (TG).
Fig. 1. Mean body weights of female and male rats received different levels of PQQ disodium salt for 13 weeks.
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2). On day 46, compared with the control group, the percentage of granulocyte was significantly higher for females at 200 mg/kg bw/ day group, and blood glucose level was significantly lower for male rats at 100 mg/kg bw/day group. No statistically significant differences were observed between the treatment groups and the control group for all other hematology and serum biochemistry parameters. 3.4. Organ weights and pathology
Fig. 2. Mean food consumption of female and male rats received different levels of PQQ disodium salt for 13 weeks.
There were no statistically significant differences in all absolute organ weights and relative organ weights (organ-to-body weight ratios) between treatment groups and the control group for male and females (Table 3). No macroscopic pathology findings were observed in all males and females. Histopathological findings are summarized in Table 4. Histopathological findings showed various lesions, including slightly sporadic focal necrosis and spotty necrosis in liver, focal necrosis in heart, deposition of calcium salts in renal tubular in kidneys and testicular atrophy in testes. The incidences and/or severities of these changes did not differ between the controls and the PQQ disodium salt treatment groups. There were no histopathological lesions observed in the cecum, colon, duodenum, esophagus, femur with bone marrow, ileum, jejunum, lacrimal
Table 1 Hematological and serum biochemical values of rats treated orally with PQQ disodium salt for 13 weeks on day 46. Parameters
WBC (×109/L) RBC (×1012/L) PLT (×109/L) HGB (g/L) LYM% (%) GR% (%) MO% (%) ALT (U/L) AST (U/L) ALP (U/L) BUN (mmol/L) CRE (μmol/L) CHO (mmol/L) TG (mmol/L) GLU (mmol/L) TP (g/L) ALB (g/L)
Females
Males
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
6.57 ± 1.79 6.97 ± 0.46 681.8 ± 116.1 151.3 ± 8.4 74.4 ± 3.2 16.1 ± 2.8 9.5 ± 1.6 35.4 ± 5.7 141.9 ± 18.2 100.2 ± 23.7 5.37 ± 1.61 61.9 ± 6.4 2.04 ± 0.42 0.44 ± 0.03 5.55 ± 0.59 68.0 ± 3.2 44.8 ± 1.7
7.30 ± 1.98 6.95 ± 0.39 678.9 ± 138.9 154.2 ± 7.4 73.4 ± 3.4 18.5 ± 2.9 8.1 ± 0.9 40.7 ± 6.6 151.0 ± 23.6 119.5 ± 57.5 5.09 ± 1.32 62.4 ± 8.7 2.07 ± 0.43 0.49 ± 0.06 6.20 ± 0.72 69.6 ± 4.3 45.6 ± 2.2
8.68 ± 2.62 6.60 ± 0.27 654.2 ± 81.9 144.8 ± 5.4 71.2 ± 3.1 21.0 ± 2.4* 7.8 ± 1.7 42.5 ± 8.9 141.3 ± 20.1 124.2 ± 49.6 6.05 ± 1.68 62.9 ± 7.0 2.04 ± 0.45 0.56 ± 0.16 6.08 ± 0.94 69.3 ± 4.9 46.0 ± 3.0
7.20 ± 1.72 7.14 ± 0.28 622.8 ± 84.6 153.5 ± 8.6 72.3 ± 6.7 18.9 ± 5.3 8.8 ± 2.4 37.4 ± 4.4 154.3 ± 22.4 90.1 ± 31.7 4.62 ± 0.80 59.8 ± 7.0 2.05 ± 0.52 0.54 ± 0.09 5.87 ± 1.00 70.5 ± 3.4 46.5 ± 1.4
8.55 ± 2.05 7.12 ± 0.31 846.6 ± 139.9 156.0 ± 5.7 73.0 ± 2.4 18.6 ± 2.3 8.4 ± 1.7 39.3 ± 2.8 180.2 ± 19.3 154.2 ± 47.7 4.42 ± 0.94 58.1 ± 6.4 1.72 ± 0.38 0.51 ± 0.09 5.27 ± 0.41 67.2 ± 4.1 43.3 ± 2.1
9.00 ± 2.00 7.09 ± 0.41 793.8 ± 201.1 156.1 ± 4.5 74.7 ± 4.4 16.0 ± 3.7 9.3 ± 2.6 38.8 ± 3.2 186.0 ± 30.1 138.0 ± 37.5 4.57 ± 1.03 57.1 ± 4.6 1.77 ± 0.38 0.59 ± 0.14 4.49 ± 0.31* 66.2 ± 4.2 43.6 ± 2.3
8.74 ± 1.90 7.24 ± 0.23 853.2 ± 138.9 165.0 ± 11.7 77.2 ± 4.2 15.5 ± 4.0 7.3 ± 1.7 39.7 ± 4.1 184.9 ± 23.4 157.8 ± 51.8 4.19 ± 0.56 61.8 ± 5.2 2.00 ± 0.39 0.54 ± 0.09 5.27 ± 1.01 69.1 ± 5.3 44.8 ± 2.1
9.11 ± 2.47 7.24 ± 0.25 730.1 ± 137.1 160.7 ± 6.7 76.2 ± 3.0 16.2 ± 3.6 7.6 ± 1.5 39.0 ± 3.9 191.2 ± 13.5 132.1 ± 36.4 4.94 ± 0.78 59.1 ± 5.9 1.74 ± 0.47 0.52 ± 0.09 5.00 ± 0.47 67.5 ± 5.1 44.4 ± 2.1
* P < 0.05 as compared with the control group.
Table 2 Hematological and serum biochemical values of rats treated orally with PQQ disodium salt for 13 weeks on day 91. Parameters
9/L)
WBC (×10 RBC (×1012/L) PLT (×109/L) HGB (g/L) LYM%(%) GR%(%) MO%(%) ALT (U/L) AST (U/L) ALP (U/L) BUN (mmol/L) CRE (μmol/L) CHO (mmol/L) TG (mmol/L) GLU (mmol/L) TP (g/L) ALB (g/L)
Females
Males
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
7.26 ± 0.80 7.38 ± 0.31 461.6 ± 65.8 167.6 ± 7.4 73.9 ± 5.3 16.2 ± 4.6 9.9 ± 2.0 20.7 ± 4.3 147.2 ± 18.1 65.0 ± 18.0 5.25 ± 1.12 64.0 ± 3.1 1.96 ± 0.47 0.64 ± 0.16 4.44 ± 1.05 78.2 ± 4.2 44.9 ± 2.5
8.17 ± 1.18 7.40 ± 0.30 444.0 ± 69.4 160.3 ± 8.4 76.3 ± 2.6 16.2 ± 2.4 7.5 ± 1.0 20.4 ± 8.2 157.6 ± 24.3 60.0 ± 10.8 5.19 ± 0.68 64.7 ± 7.5 2.07 ± 0.45 0.52 ± 0.05 4.94 ± 0.60 77.9 ± 3.7 45.1 ± 2.1
7.50 ± 2.14 7.19 ± 0.34 467.3 ± 68.2 161.0 ± 9.6 71.6 ± 4.6 18.5 ± 1.9 9.9 ± 4.4 18.1 ± 5.7 159.7 ± 19.9 63.1 ± 13.8 5.46 ± 0.79 68.3 ± 3.4 1.82 ± 0.51 0.53 ± 0.13 4.91 ± 0.79 78.2 ± 3.0 45.1 ± 2.2
7.28 ± 1.40 7.26 ± 0.63 408.5 ± 67.3 156.6 ± 12.6 73.9 ± 3.5 17.7 ± 2.5 8.4 ± 2.1 19.8 ± 3.2 156.7 ± 20.7 71.0 ± 24.6 5.57 ± 1.17 67.9 ± 5.5 2.11 ± 0.64 0.57 ± 0.13 4.62 ± 0.59 79.3 ± 1.8 45.9 ± 1.9
10.60 ± 0.95 7.33 ± 0.26 656.8 ± 145.9 168.0 ± 17.5 73.1 ± 1.4 19.6 ± 1.7 7.3 ± 1.2 29.5 ± 9.6 171.4 ± 36.8 131.3 ± 22.6 5.88 ± 1.05 67.6 ± 5.9 1.89 ± 0.34 0.84 ± 0.30 4.39 ± 0.56 74.5 ± 3.3 41.7 ± 2.2
11.52 ± 2.75 7.41 ± 0.87 597.0 ± 62.7 159.2 ± 13.4 74.3 ± 2.7 17.4 ± 3.3 8.3 ± 2.1 28.2 ± 6.7 194.1 ± 32.6 134.2 ± 25.2 5.03 ± 0.62 62.9 ± 6.6 1.65 ± 0.25 0.77 ± 0.23 4.84 ± 0.53 71.9 ± 3.6 40.5 ± 1.5
9.86 ± 1.95 7.52 ± 0.40 625.3 ± 97.8 162.0 ± 13.0 71.1 ± 4.2 20.3 ± 2.7 8.6 ± 2.1 24.6 ± 4.3 174.1 ± 18.8 117.9 ± 27.2 5.03 ± 0.61 66.1 ± 6.4 1.77 ± 0.35 0.59 ± 0.13 4.76 ± 0.53 75.8 ± 3.4 42.1 ± 1.0
10.71 ± 2.88 7.42 ± 0.39 553.3 ± 83.5 159.1 ± 6.2 73.0 ± 4.0 18.7 ± 2.5 8.3 ± 2.1 27.8 ± 9.4 173.2 ± 17.5 123.8 ± 19.8 5.53 ± 1.16 68.5 ± 5.6 1.69 ± 0.38 0.66 ± 0.22 4.66 ± 0.46 74.2 ± 2.7 41.3 ± 0.9
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Table 3 Absolute and relative organ weights of rats treated orally with PQQ disodium salt for 13 weeks. Parameters
Liver (g) Kidneys (g) Spleen (g) Thymus (g) Heart (g) Testes (g)
Females
Males
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
8.01 ± 0.71 2.17 ± 0.18 0.55 ± 0.07 0.29 ± 0.09 1.04 ± 0.12 –
7.79 ± 0.89 2.24 ± 0.22 0.49 ± 0.07 0.31 ± 0.10 0.97 ± 0.07 –
7.86 ± 1.17 2.26 ± 0.20 0.58 ± 0.14 0.31 ± 0.11 0.99 ± 0.11 –
7.91 ± 1.31 2.31 ± 0.16 0.56 ± 0.11 0.29 ± 0.08 1.03 ± 0.14 –
11.49 ± 1.16 3.29 ± 0.42 0.71 ± 0.16 0.38 ± 0.05 1.47 ± 0.13 3.35 ± 0.18
11.16 ± 1.40 3.31 ± 0.34 0.72 ± 0.10 0.42 ± 0.10 1.50 ± 0.19 3.30 ± 0.33
11.79 ± 1.65 3.59 ± 0.44 0.68 ± 0.10 0.34 ± 0.07 1.52 ± 0.15 3.54 ± 0.31
12.38 ± 1.66 3.67 ± 0.74 0.69 ± 0.12 0.36 ± 0.07 1.49 ± 0.20 3.16 ± 0.29
2.97 ± 0.31 0.86 ± 0.07 0.22 ± 0.05 0.12 ± 0.04 0.38 ± 0.02 –
2.94 ± 0.42 0.86 ± 0.09 0.21 ± 0.04 0.11 ± 0.03 0.38 ± 0.05 –
2.61 ± 0.43 0.75 ± 0.15 0.16 ± 0.04 0.08 ± 0.01 0.33 ± 0.06 0.76 ± 0.09
2.46 ± 0.14 0.73 ± 0.06 0.16 ± 0.02 0.09 ± 0.02 0.33 ± 0.03 0.73 ± 0.10
2.60 ± 0.31 0.80 ± 0.14 0.15 ± 0.02 0.07 ± 0.01 0.34 ± 0.06 0.78 ± 0.06
2.74 ± 0.26 0.82 ± 0.18 0.15 ± 0.03 0.08 ± 0.01 0.33 ± 0.05 0.70 ± 0.09
Organ-to-body weight ratios (relative organ weights) Liver (%) Kidneys (%) Spleen (%) Thymus (%) Heart (%) Testes (%)
3.02 ± 0.22 0.82 ± 0.06 0.21 ± 0.03 0.11 ± 0.04 0.39 ± 0.03 –
3.00 ± 0.36 0.86 ± 0.10 0.19 ± 0.04 0.12 ± 0.04 0.37 ± 0.02 –
–, Not applicable.
Table 4 Histopathological changes of rats treated orally with PQQ disodium salt for 13 weeks. Organs
Liver Heart Kidneys Testes a
Lesions
Slight sporadic focal necrosis Spotty necrosis Focal necrosis Deposition of calcium salts in renal tubular Testicular atrophy
Dosea 0 mg/kg BW
100 mg/kg BW
200 mg/kg BW
400 mg/kg BW
0 3 3 0
0 1 1 0
2 3 2 0
0 1 5 1
0
0
0
1
10 animals/sex/group.
gland, lung, lymph node, mammary gland, nasal turbinates, pancreas, pituitary gland, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin, spinal cord, spleen, sternum with bone marrow, thymus, trachea, urinary bladder, vagina, ovary and adrenals. 4. Discussion Previous studies have shown that PQQ has various benefits on physiological and biochemical processes and plays an important nutritional role in rodents, such as improvements in growth, reproduction performance, immune function, cardioprotection and neuroprotection (Steinberg et al., 1994, 2003; Tao et al., 2007; Zhang et al., 2006). However PQQ cannot be synthesized in higher organisms, and so has a potential to be used as a dietary supplement. In a recent report, the safety of PQQ disodium salt has been demonstrated in a 13-week subchronic oral toxicity study, the no-observedadverse-effect level (NOAEL) in rats was 100 mg/kg bw/day (Nakano et al., 2014). In this study, we demonstrate the safety of PQQ disodium salt by performing a 13-week subchronic oral study in SD rats up to a level of 400 mg/kg bw/day. In our present study, no PQQ disodium salt-related deaths or abnormalities in clinical signs were noted. No significant changes in body weight, food consumption, necropsy findings were observed in relation to the administration of PQQ disodium salt by gavage at concentrations as high as 400 mg/kg bw/day. In hematology and serum biochemistry analyses, isolated statistically significant changes of some parameters in PQQ disodium salt treatment groups were observed. Specifically, the significant elevation of the percentage of granulocyte in females at 200 mg/kg bw/day group and the decrease of the level of blood glucose in males at 100 mg/kg bw/day group were noticed, as compared with the control group. However, the changes were not dose-related and were
within the laboratory’s historical normal range of controls; therefore, the effects were not considered to be toxicological significance. In the histopathological examination, some microscopic changes in the liver, heart, kidneys and testes were noted. However, all of those pathological changes were within the range of normal background lesions and sporadically detected in control and treatment groups; therefore, these effects could be considered incidental and reflected the usual individual variability without any relationship to the treatment. In conclusion, the results of our present study demonstrate that the NOAEL for PQQ disodium salt was 400 mg/kg bw/day in male and female rats, the highest dose tested. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. References Felton, L.M., Anthony, C., 2005. Biochemistry: role of PQQ as a mammalian enzyme cofactor. Nature 433 (7025), E10, discussion E11–E12. Gu, J., Yun, X., Chen, X., Zhang, S., Yang, J., Wu, Y., 2012. Purified pyrroloquinoline quinone fortified food. US patent 20120070866 A1. Kasahara, T., Kato, T., 2003. Nutritional biochemistry: a new redox-cofactor vitamin for mammals. Nature 422 (6934), 832. Kumazawa, T., Seno, H., Urakami, T., Matsumoto, T., Suzuki, O., 1992. Trace levels of pyrroloquinoline quinone in human and rat samples detected by gas chromatography/mass spectrometry. Biochim. Biophys. Acta 1156 (1), 62–66. Kumazawa, T., Sato, K., Seno, H., Ishii, A., Suzuki, O., 1995. Levels of pyrroloquinoline quinone in various foods. Biochem. J. 307, 331–333.
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Steinberg, F.M., Gershwin, M.E., Rucker, R.B., 1994. Dietary pyrroloquinoline quinone: growth and immune response in BALB/c mice. J. Nutr. 124 (5), 744–753. Sumi, M., Ogino, F., Kamiya, T., Nakano, M., Kikkawa, K., 2012. Insulin resistance improving agent. US patent 8097635 B2. Tao, R., Karliner, J.S., Simonis, U., Zheng, J., Zhang, J., Honbo, N., et al., 2007. Pyrroloquinoline quinone preserves mitochondrial function and prevents oxidative injury in adult rat cardiac myocytes. Biochem. Biophys. Res. Commun. 363 (2), 257–262. Tsuji, T., Yamaguchi, K., Kondo, K., Urakami, T., 1998. Nerve growth factor production accelerators and compositions for preventing or treating neuronal degeneration. US patent 5846977 A. US Food and Drug Administration (FDA), 2007. Subchronic toxicity studies with rodents. In: Guidance for Industry and Other Stakeholders, Toxicological Principles for the Safety Assessment of Food Ingredients Redbook 2000. FDA, Maryland, USA. Watanabe, A., Hobara, N., Ohsawa, T., Higashi, T., Tsuji, T., 1989. Nephrotoxicity of pyrroloquinoline quinone in rats. Hiroshima J. Med. Sci. 38 (1), 49–51. Zhang, Y., Feustel, P.J., Kimelberg, H.K., 2006. Neuroprotection by pyrroloquinoline quinone (PQQ) in reversible middle cerebral artery occlusion in the adult rat. Brain Res. 1094 (1), 200–206. Zhou, L., Yun, X., Gu, J., 2010. Application of pyrro-quinoline quinine in treating and preventing atherosclerosis. CN patent 101756984.
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
A subchronic oral toxicity study on pyrroloquinoline quinone (PQQ) disodium salt in rats Chunlai Liang, Xin Zhang, Wei Wang, Yan Song, 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
I N F O
Article history: Received 4 June 2014 Accepted 7 November 2014 Available online 15 November 2014 Keywords: Pyrroloquinoline quinone disodium salt Subchronic toxicity Rats NOAEL
A B S T R A C T
A subchronic oral toxicity study on pyrroloquinoline quinone (PQQ) disodium salt was performed in rats. Sprague-Dawley rats were randomly divided into four groups (10 rats/sex/group) and administered with PQQ disodium salt at doses of 0 (control), 100, 200 and 400 mg/kg bw/day by gavage for 13 weeks. Daily clinical observations and weekly measurement of body weights and food consumption were conducted. Blood samples were obtained on day 46 and day 91 for measurement of hematology and serum biochemical parameters. Animals were euthanized for necropsy, selected organs were weighted and recorded. Histological examination was performed on all tissues from animals in the control and PQQ disodium salt treatment groups. No mortality or toxicologically significant changes in clinical signs, body weight, food consumption, necropsy findings or organ weights was observed. Differences between treated and control groups in some hematological and serum biochemical examinations and histopathological examination were not considered treatment-related. The no-observed-adverse-effect-level (NOAEL) of PQQ disodium salt in rats was considered to be 400 mg/kg bw/day for both sexes, the highest dose tested. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction In 1979, pyrroloquinoline quinone (PQQ) was identified as a novel coenzyme in several bacterial dehydrogenases (Salisbury et al., 1979). Since then, it has been detected in various foods and in tissues and body fluids of humans and rats (Kumazawa et al., 1992, 1995; Noji et al., 2007). PQQ can be synthesized by some microorganisms, the major source of PQQ in human is believed to be microbial sources from the diet (Kumazawa et al., 1995; Misra et al., 2012). PQQ plays an important nutritional role in both bacteria and higher organisms and is involved in numerous physiological and biochemical processes (Misra et al., 2012; Rucker et al., 2009). It has been reported to have benefits in rodents related to improvement of neonatal growth, reproductive performance and immune function, as well as cardioprotection and neuroprotection (Steinberg et al., 1994, 2003; Tao et al., 2007; Zhang et al., 2006).
Abbreviations: ALB, albumin; ALT, alanine aminotransferase; ALP, alkaline phosphatase; ANOVA, analysis of variance; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CHO, cholesterol; CRE, creatinine; FDA, Food and Drug Administration; GLU, glucose; GR, granulocyte; HGB, hemoglobin; LYM, lymphocyte; MO, monocyte; NOAEL, no-observed-adverse-effect-level; PLT, platelet count; PQQ, pyrroloquinoline quinone; RBC, red blood cell count; SD rat, Sprague-Dawley rat; TG, triglyceride; TP, total protein; WBC, white blood cell count. * Corresponding author. Key Laboratory of Food Safety Risk Assessment of Ministry of Health, National Center for Food Safety Risk Assessment, Beijing 100021, China. Tel./fax: +86-10-67770977. E-mail address: [email protected] (X. Jia). http://dx.doi.org/10.1016/j.fct.2014.11.005 0278-6915/© 2014 Elsevier Ltd. All rights reserved.
In 2003, two scientists identified a PQQ-dependent dehydrogenase enzyme in mice and found that PQQ acted as a mammalian redox cofactor in lysine metabolism. Thus they concluded PQQ to be a newcomer to the B group of vitamins (Kasahara and Kato, 2003). However, the statement was subsequently doubted by other scientists who claimed that sufficient evidence was not available to conclude that PQQ performed an essential vitamin function in mammals (Felton and Anthony, 2005; Rucker et al., 2005). Nevertheless, PQQ did appear to have growth promoting properties in rodents and there are several patents on PQQ applications in different countries including the United States of America, Japan and China focusing on its commercial potential as human food and/or supplement ingredient (Gu et al., 2012; Sumi et al., 2012; Tsuji et al., 1998; Zhou et al., 2010). For any new dietary ingredient intended to be added into foods or used as a dietary supplement, it is necessary and essential to conduct a safety assessment, especially a toxicological evaluation. However, there are only a few reports on the toxicity evaluation of PQQ. One study evaluated the potential genotoxicity of PQQ disodium salt using a battery of genotoxicity tests (Ames test, in vitro chromosomal aberration test in Chinese hamster lung cells, in vitro chromosomal aberration test in human peripheral blood lymphocytes and in vivo micronucleus assay in mice) and concluded that PQQ had no genotoxic activity (Nakano et al., 2013). In another study, functional and morphologic changes of kidneys were observed when PQQ was intraperitoneally injected into rats at a dose of 11.5 mg/ kg (Watanabe et al., 1989). Subchronic toxicity studies can provide more information on the possible health hazards of test
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substances from repeated exposure over a prolonged period of time; therefore, a 13-week subchronic oral toxicity test on PQQ disodium salt was conducted in rats in this study. 2. Materials and methods 2.1. Test substance PQQ disodium salt, molecular weight 374.17, water-soluble reddish brown crystalline powder with mild hydroscopic property, purity >98%, was provided by Shanghai Med Co., Ltd., China.
2.2. Animals Weanling Sprague-Dawley (SD) rats, specific pathogen-free grade, were purchased from Vital River Laboratory Animal Technology Co, Ltd (Beijing, China). Body weights of rats at receipt were 60–80 g. All animals were examined for clinical signs of ill health on receipt and observed within 5 days of arrival. Rats were housed in an environmental controlled room with the temperature at 23 ± 2 °C and the relative humidity within the range of 40–70%. Air was changed 10–15 times per hour. Light was set for a 12 hr light/dark cycle. Rats were individually housed in suspended stainless steel, open-mesh cages and allowed free access to pellet feed and tap water during the experiments.
2.3. Experimental design Eighty healthy SD rats were randomly divided into four groups, 10 animals/sex/ group. One group administered with water by gavage served as the control. An acute oral toxicity of PQQ disodium salt was previously conducted by Horn’s method as described in the Chinese Toxicology Assessment Procedures and Methods for Food Safety (Chinese Standard GB15193.3-2003) in our lab, the dose selection for this study was based on the result of the acute oral toxicity (the LD50 of females and males was 5.01 and 3.69 g/kg, respectively), 400 mg/kg bw/day (around 10% of LD50) was selected as the high dose level, and 200 mg/kg bw/day and 100 mg/kg bw/day were chosen as the other two lower levels. PQQ disodium salt was administered to rats by gavage once daily for 13 weeks. Clinical observations were recorded daily. Body weights were measured weekly and the gavage volume (1 ml/100 g body weight) was adjusted accordingly. Blood samples were obtained in the middle of the study (day 46) and at the end of the study for measurement of hematology and serum biochemistry parameters. At the end of the study, all animals were euthanized for necropsy. Selected organs were weighted and recorded. Histological examination was performed on all tissues from animals in the control group and PQQ disodium salt treatment groups. The study was 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 (US Food and Drug Administration (FDA), 2007). The protocol has been approved by the Office of Laboratory Animal Welfare, National Institute for Nutrition and Food Safety.
2.4. Clinical observations
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2.7. Pathology All rats were humanely sacrificed at the end of the test, and a complete necropsy was performed including an examination of the external features of the carcass, external body orifices, the abdominal, thoracic, and cranial cavities, organs and tissues. Organ weights were obtained for the heart, kidneys, liver, spleen, testes and thymus. Paired organs were weighed together. Organ-to-body weight ratios (relative organ weights) were also calculated. In addition to the above-mentioned organs, the following tissues (when present) were sampled and fixed in 10% neutral-buffered formalin: cecum, colon, duodenum, esophagus, femur with bone marrow, ileum, jejunum, lacrimal gland, lung, lymph node, mammary gland, nasal turbinates, pancreas, pituitary gland, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin, spinal cord, sternum with bone marrow, trachea, urinary bladder, vagina, ovary and adrenals. All stored organs and tissues from each animal in the control group and PQQ disodium salt treatment groups were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and subjected to microscopic examination. Macroscopic lesions observed at necropsy were also examined from each animal in all groups. 2.8. Statistical analysis Data were presented as mean ± SD. The SPSS Statistical System (SPSS for Windows 18.0, Chicago, USA) was used to analyze body weights, food consumption, hematology and serum biochemistry parameters, and organ weights, followed by testing for variance homogeneity. Homogeneous data were analyzed using the analysis of variance (ANOVA) and the significance of differences between control and treated groups were analyzed using Dunnett’s multiple comparisons. P < 0.05 was considered statistically significant.
3. Results 3.1. Mortality and clinical signs No mortality or treatment related adverse clinical reactions were found during the study. 3.2. Body weight and food consumption There were no statistically significant differences in body weights between the treatment groups and the control group in each week (Fig. 1). No significant differences were observed between the treatment groups and the control group for weekly food consumption of females and males (Fig. 2). 3.3. Hematology and serum biochemistry There were some sporadic, statistically significant changes in some hematology and serum biochemical parameters (Tables 1 and
Each animal was observed twice daily for abnormalities, physical appearance and mortality throughout the study. Observations included, but were not limited to, changes in skin, fur, eyes, appearance, salivary gland secretions, oral mucosa, fecal characteristics, respiration and behavior.
2.5. Body weight and food consumption The body weights of rats were measured pre-test, weekly thereafter and at sacrifice after fasting. Food consumption was measured once a week during the experiment period.
2.6. Hematology and serum biochemistry On day 46 and day 91, rats were anesthetized with 3% sodium pentobarbital solution and blood samples were collected from the tail vein. Blood samples for hematology were collected into tubes containing ethylenediaminetetraacetic acid anticoagulant. A COULTER Ac.T diff2 Hematology Analyzer (Beckman Coulter Corporation) was employed to measure the following parameters: red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), white blood cell count (WBC) and WBC differential count of lymphocyte (LYM), granulocyte (GR) and monocyte (MO). Blood samples for serum biochemistry were collected into tubes containing no anticoagulant and centrifuged to obtain serum. An automatic analyzer (Hitachi 7080, Hitachi High-Technologies Corporation) was used to analyze the following parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), glucose (GLU), blood urea nitrogen (BUN), creatinine (CRE), cholesterol (CHO) and triglyceride (TG).
Fig. 1. Mean body weights of female and male rats received different levels of PQQ disodium salt for 13 weeks.
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2). On day 46, compared with the control group, the percentage of granulocyte was significantly higher for females at 200 mg/kg bw/ day group, and blood glucose level was significantly lower for male rats at 100 mg/kg bw/day group. No statistically significant differences were observed between the treatment groups and the control group for all other hematology and serum biochemistry parameters. 3.4. Organ weights and pathology
Fig. 2. Mean food consumption of female and male rats received different levels of PQQ disodium salt for 13 weeks.
There were no statistically significant differences in all absolute organ weights and relative organ weights (organ-to-body weight ratios) between treatment groups and the control group for male and females (Table 3). No macroscopic pathology findings were observed in all males and females. Histopathological findings are summarized in Table 4. Histopathological findings showed various lesions, including slightly sporadic focal necrosis and spotty necrosis in liver, focal necrosis in heart, deposition of calcium salts in renal tubular in kidneys and testicular atrophy in testes. The incidences and/or severities of these changes did not differ between the controls and the PQQ disodium salt treatment groups. There were no histopathological lesions observed in the cecum, colon, duodenum, esophagus, femur with bone marrow, ileum, jejunum, lacrimal
Table 1 Hematological and serum biochemical values of rats treated orally with PQQ disodium salt for 13 weeks on day 46. Parameters
WBC (×109/L) RBC (×1012/L) PLT (×109/L) HGB (g/L) LYM% (%) GR% (%) MO% (%) ALT (U/L) AST (U/L) ALP (U/L) BUN (mmol/L) CRE (μmol/L) CHO (mmol/L) TG (mmol/L) GLU (mmol/L) TP (g/L) ALB (g/L)
Females
Males
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
6.57 ± 1.79 6.97 ± 0.46 681.8 ± 116.1 151.3 ± 8.4 74.4 ± 3.2 16.1 ± 2.8 9.5 ± 1.6 35.4 ± 5.7 141.9 ± 18.2 100.2 ± 23.7 5.37 ± 1.61 61.9 ± 6.4 2.04 ± 0.42 0.44 ± 0.03 5.55 ± 0.59 68.0 ± 3.2 44.8 ± 1.7
7.30 ± 1.98 6.95 ± 0.39 678.9 ± 138.9 154.2 ± 7.4 73.4 ± 3.4 18.5 ± 2.9 8.1 ± 0.9 40.7 ± 6.6 151.0 ± 23.6 119.5 ± 57.5 5.09 ± 1.32 62.4 ± 8.7 2.07 ± 0.43 0.49 ± 0.06 6.20 ± 0.72 69.6 ± 4.3 45.6 ± 2.2
8.68 ± 2.62 6.60 ± 0.27 654.2 ± 81.9 144.8 ± 5.4 71.2 ± 3.1 21.0 ± 2.4* 7.8 ± 1.7 42.5 ± 8.9 141.3 ± 20.1 124.2 ± 49.6 6.05 ± 1.68 62.9 ± 7.0 2.04 ± 0.45 0.56 ± 0.16 6.08 ± 0.94 69.3 ± 4.9 46.0 ± 3.0
7.20 ± 1.72 7.14 ± 0.28 622.8 ± 84.6 153.5 ± 8.6 72.3 ± 6.7 18.9 ± 5.3 8.8 ± 2.4 37.4 ± 4.4 154.3 ± 22.4 90.1 ± 31.7 4.62 ± 0.80 59.8 ± 7.0 2.05 ± 0.52 0.54 ± 0.09 5.87 ± 1.00 70.5 ± 3.4 46.5 ± 1.4
8.55 ± 2.05 7.12 ± 0.31 846.6 ± 139.9 156.0 ± 5.7 73.0 ± 2.4 18.6 ± 2.3 8.4 ± 1.7 39.3 ± 2.8 180.2 ± 19.3 154.2 ± 47.7 4.42 ± 0.94 58.1 ± 6.4 1.72 ± 0.38 0.51 ± 0.09 5.27 ± 0.41 67.2 ± 4.1 43.3 ± 2.1
9.00 ± 2.00 7.09 ± 0.41 793.8 ± 201.1 156.1 ± 4.5 74.7 ± 4.4 16.0 ± 3.7 9.3 ± 2.6 38.8 ± 3.2 186.0 ± 30.1 138.0 ± 37.5 4.57 ± 1.03 57.1 ± 4.6 1.77 ± 0.38 0.59 ± 0.14 4.49 ± 0.31* 66.2 ± 4.2 43.6 ± 2.3
8.74 ± 1.90 7.24 ± 0.23 853.2 ± 138.9 165.0 ± 11.7 77.2 ± 4.2 15.5 ± 4.0 7.3 ± 1.7 39.7 ± 4.1 184.9 ± 23.4 157.8 ± 51.8 4.19 ± 0.56 61.8 ± 5.2 2.00 ± 0.39 0.54 ± 0.09 5.27 ± 1.01 69.1 ± 5.3 44.8 ± 2.1
9.11 ± 2.47 7.24 ± 0.25 730.1 ± 137.1 160.7 ± 6.7 76.2 ± 3.0 16.2 ± 3.6 7.6 ± 1.5 39.0 ± 3.9 191.2 ± 13.5 132.1 ± 36.4 4.94 ± 0.78 59.1 ± 5.9 1.74 ± 0.47 0.52 ± 0.09 5.00 ± 0.47 67.5 ± 5.1 44.4 ± 2.1
* P < 0.05 as compared with the control group.
Table 2 Hematological and serum biochemical values of rats treated orally with PQQ disodium salt for 13 weeks on day 91. Parameters
9/L)
WBC (×10 RBC (×1012/L) PLT (×109/L) HGB (g/L) LYM%(%) GR%(%) MO%(%) ALT (U/L) AST (U/L) ALP (U/L) BUN (mmol/L) CRE (μmol/L) CHO (mmol/L) TG (mmol/L) GLU (mmol/L) TP (g/L) ALB (g/L)
Females
Males
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
7.26 ± 0.80 7.38 ± 0.31 461.6 ± 65.8 167.6 ± 7.4 73.9 ± 5.3 16.2 ± 4.6 9.9 ± 2.0 20.7 ± 4.3 147.2 ± 18.1 65.0 ± 18.0 5.25 ± 1.12 64.0 ± 3.1 1.96 ± 0.47 0.64 ± 0.16 4.44 ± 1.05 78.2 ± 4.2 44.9 ± 2.5
8.17 ± 1.18 7.40 ± 0.30 444.0 ± 69.4 160.3 ± 8.4 76.3 ± 2.6 16.2 ± 2.4 7.5 ± 1.0 20.4 ± 8.2 157.6 ± 24.3 60.0 ± 10.8 5.19 ± 0.68 64.7 ± 7.5 2.07 ± 0.45 0.52 ± 0.05 4.94 ± 0.60 77.9 ± 3.7 45.1 ± 2.1
7.50 ± 2.14 7.19 ± 0.34 467.3 ± 68.2 161.0 ± 9.6 71.6 ± 4.6 18.5 ± 1.9 9.9 ± 4.4 18.1 ± 5.7 159.7 ± 19.9 63.1 ± 13.8 5.46 ± 0.79 68.3 ± 3.4 1.82 ± 0.51 0.53 ± 0.13 4.91 ± 0.79 78.2 ± 3.0 45.1 ± 2.2
7.28 ± 1.40 7.26 ± 0.63 408.5 ± 67.3 156.6 ± 12.6 73.9 ± 3.5 17.7 ± 2.5 8.4 ± 2.1 19.8 ± 3.2 156.7 ± 20.7 71.0 ± 24.6 5.57 ± 1.17 67.9 ± 5.5 2.11 ± 0.64 0.57 ± 0.13 4.62 ± 0.59 79.3 ± 1.8 45.9 ± 1.9
10.60 ± 0.95 7.33 ± 0.26 656.8 ± 145.9 168.0 ± 17.5 73.1 ± 1.4 19.6 ± 1.7 7.3 ± 1.2 29.5 ± 9.6 171.4 ± 36.8 131.3 ± 22.6 5.88 ± 1.05 67.6 ± 5.9 1.89 ± 0.34 0.84 ± 0.30 4.39 ± 0.56 74.5 ± 3.3 41.7 ± 2.2
11.52 ± 2.75 7.41 ± 0.87 597.0 ± 62.7 159.2 ± 13.4 74.3 ± 2.7 17.4 ± 3.3 8.3 ± 2.1 28.2 ± 6.7 194.1 ± 32.6 134.2 ± 25.2 5.03 ± 0.62 62.9 ± 6.6 1.65 ± 0.25 0.77 ± 0.23 4.84 ± 0.53 71.9 ± 3.6 40.5 ± 1.5
9.86 ± 1.95 7.52 ± 0.40 625.3 ± 97.8 162.0 ± 13.0 71.1 ± 4.2 20.3 ± 2.7 8.6 ± 2.1 24.6 ± 4.3 174.1 ± 18.8 117.9 ± 27.2 5.03 ± 0.61 66.1 ± 6.4 1.77 ± 0.35 0.59 ± 0.13 4.76 ± 0.53 75.8 ± 3.4 42.1 ± 1.0
10.71 ± 2.88 7.42 ± 0.39 553.3 ± 83.5 159.1 ± 6.2 73.0 ± 4.0 18.7 ± 2.5 8.3 ± 2.1 27.8 ± 9.4 173.2 ± 17.5 123.8 ± 19.8 5.53 ± 1.16 68.5 ± 5.6 1.69 ± 0.38 0.66 ± 0.22 4.66 ± 0.46 74.2 ± 2.7 41.3 ± 0.9
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Table 3 Absolute and relative organ weights of rats treated orally with PQQ disodium salt for 13 weeks. Parameters
Liver (g) Kidneys (g) Spleen (g) Thymus (g) Heart (g) Testes (g)
Females
Males
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
0 mg/kgbw
100 mg/kgbw
200 mg/kgbw
400 mg/kgbw
8.01 ± 0.71 2.17 ± 0.18 0.55 ± 0.07 0.29 ± 0.09 1.04 ± 0.12 –
7.79 ± 0.89 2.24 ± 0.22 0.49 ± 0.07 0.31 ± 0.10 0.97 ± 0.07 –
7.86 ± 1.17 2.26 ± 0.20 0.58 ± 0.14 0.31 ± 0.11 0.99 ± 0.11 –
7.91 ± 1.31 2.31 ± 0.16 0.56 ± 0.11 0.29 ± 0.08 1.03 ± 0.14 –
11.49 ± 1.16 3.29 ± 0.42 0.71 ± 0.16 0.38 ± 0.05 1.47 ± 0.13 3.35 ± 0.18
11.16 ± 1.40 3.31 ± 0.34 0.72 ± 0.10 0.42 ± 0.10 1.50 ± 0.19 3.30 ± 0.33
11.79 ± 1.65 3.59 ± 0.44 0.68 ± 0.10 0.34 ± 0.07 1.52 ± 0.15 3.54 ± 0.31
12.38 ± 1.66 3.67 ± 0.74 0.69 ± 0.12 0.36 ± 0.07 1.49 ± 0.20 3.16 ± 0.29
2.97 ± 0.31 0.86 ± 0.07 0.22 ± 0.05 0.12 ± 0.04 0.38 ± 0.02 –
2.94 ± 0.42 0.86 ± 0.09 0.21 ± 0.04 0.11 ± 0.03 0.38 ± 0.05 –
2.61 ± 0.43 0.75 ± 0.15 0.16 ± 0.04 0.08 ± 0.01 0.33 ± 0.06 0.76 ± 0.09
2.46 ± 0.14 0.73 ± 0.06 0.16 ± 0.02 0.09 ± 0.02 0.33 ± 0.03 0.73 ± 0.10
2.60 ± 0.31 0.80 ± 0.14 0.15 ± 0.02 0.07 ± 0.01 0.34 ± 0.06 0.78 ± 0.06
2.74 ± 0.26 0.82 ± 0.18 0.15 ± 0.03 0.08 ± 0.01 0.33 ± 0.05 0.70 ± 0.09
Organ-to-body weight ratios (relative organ weights) Liver (%) Kidneys (%) Spleen (%) Thymus (%) Heart (%) Testes (%)
3.02 ± 0.22 0.82 ± 0.06 0.21 ± 0.03 0.11 ± 0.04 0.39 ± 0.03 –
3.00 ± 0.36 0.86 ± 0.10 0.19 ± 0.04 0.12 ± 0.04 0.37 ± 0.02 –
–, Not applicable.
Table 4 Histopathological changes of rats treated orally with PQQ disodium salt for 13 weeks. Organs
Liver Heart Kidneys Testes a
Lesions
Slight sporadic focal necrosis Spotty necrosis Focal necrosis Deposition of calcium salts in renal tubular Testicular atrophy
Dosea 0 mg/kg BW
100 mg/kg BW
200 mg/kg BW
400 mg/kg BW
0 3 3 0
0 1 1 0
2 3 2 0
0 1 5 1
0
0
0
1
10 animals/sex/group.
gland, lung, lymph node, mammary gland, nasal turbinates, pancreas, pituitary gland, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (thigh), skin, spinal cord, spleen, sternum with bone marrow, thymus, trachea, urinary bladder, vagina, ovary and adrenals. 4. Discussion Previous studies have shown that PQQ has various benefits on physiological and biochemical processes and plays an important nutritional role in rodents, such as improvements in growth, reproduction performance, immune function, cardioprotection and neuroprotection (Steinberg et al., 1994, 2003; Tao et al., 2007; Zhang et al., 2006). However PQQ cannot be synthesized in higher organisms, and so has a potential to be used as a dietary supplement. In a recent report, the safety of PQQ disodium salt has been demonstrated in a 13-week subchronic oral toxicity study, the no-observedadverse-effect level (NOAEL) in rats was 100 mg/kg bw/day (Nakano et al., 2014). In this study, we demonstrate the safety of PQQ disodium salt by performing a 13-week subchronic oral study in SD rats up to a level of 400 mg/kg bw/day. In our present study, no PQQ disodium salt-related deaths or abnormalities in clinical signs were noted. No significant changes in body weight, food consumption, necropsy findings were observed in relation to the administration of PQQ disodium salt by gavage at concentrations as high as 400 mg/kg bw/day. In hematology and serum biochemistry analyses, isolated statistically significant changes of some parameters in PQQ disodium salt treatment groups were observed. Specifically, the significant elevation of the percentage of granulocyte in females at 200 mg/kg bw/day group and the decrease of the level of blood glucose in males at 100 mg/kg bw/day group were noticed, as compared with the control group. However, the changes were not dose-related and were
within the laboratory’s historical normal range of controls; therefore, the effects were not considered to be toxicological significance. In the histopathological examination, some microscopic changes in the liver, heart, kidneys and testes were noted. However, all of those pathological changes were within the range of normal background lesions and sporadically detected in control and treatment groups; therefore, these effects could be considered incidental and reflected the usual individual variability without any relationship to the treatment. In conclusion, the results of our present study demonstrate that the NOAEL for PQQ disodium salt was 400 mg/kg bw/day in male and female rats, the highest dose tested. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. References Felton, L.M., Anthony, C., 2005. Biochemistry: role of PQQ as a mammalian enzyme cofactor. Nature 433 (7025), E10, discussion E11–E12. Gu, J., Yun, X., Chen, X., Zhang, S., Yang, J., Wu, Y., 2012. Purified pyrroloquinoline quinone fortified food. US patent 20120070866 A1. Kasahara, T., Kato, T., 2003. Nutritional biochemistry: a new redox-cofactor vitamin for mammals. Nature 422 (6934), 832. Kumazawa, T., Seno, H., Urakami, T., Matsumoto, T., Suzuki, O., 1992. Trace levels of pyrroloquinoline quinone in human and rat samples detected by gas chromatography/mass spectrometry. Biochim. Biophys. Acta 1156 (1), 62–66. Kumazawa, T., Sato, K., Seno, H., Ishii, A., Suzuki, O., 1995. Levels of pyrroloquinoline quinone in various foods. Biochem. J. 307, 331–333.
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