Toxicologic Assessment of Paecilomyces Tenuipes in Rats: Renal Toxicity and Mutagenic Potential

Regulatory Toxicology and Pharmacology 70 (2014) 527–534

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Regulatory Toxicology and Pharmacology

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Toxicologic assessment of Paecilomyces tenuipes in rats: Renal toxicity and mutagenic potential

Jeong-Hwan Che a,b,1, Jun-Won Yun b,1, Eun-Young Cho b, Seung-Hyun Kim b, Yun-Soon Kim b, ⇑ Woo Ho Kim c, Jae-Hak Park d, Woo-Chan Son e, Mi Kyung Kim f,g, Byeong-Cheol Kang a,b,h, a Biomedical Center for Animal Resource and Development, Bio-Max Institute, Seoul National University, Seoul, Republic of Korea b Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea c Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea d Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea e Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea f Biofood Network, Ewha Womans University, Seoul, Republic of Korea g BiofoodCRO, Seoul, Republic of Korea h Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea article info abstract

Article history: Paecilomyces tenuipes is entomogenous fungus that is called snow-ﬂake Dongchunghacho in Korea. Received 31 July 2014 Although it is widely used in traditional medicines, its safety has not yet been comprehensively investi- Available online 16 September 2014 gated. Therefore, the aim of this study was to evaluate the genotoxicity, acute and subchronic toxicity of

P. tenuipes. The acute oral LD50 of P. tenuipes extract in rats was estimated to be greater than 2000 mg/kg Keywords: of body weight. In the subchronic study, the oral treatment of rats with 500, 1000 or 2000 mg/kg Paecilomyces tenuipes P. tenuipes extract daily for 13 weeks did not induce any dose-related changes (body weight, food con- Toxicity sumption, clinical observation, urinalysis, hematology, clinical chemistry and organ weight). In contrast, Subchronic histopathological observation revealed that P. tenuipes extract induced karyomegaly in outer medulla of Genotoxicity Kidney kidney in all treated rats. Importantly, P. tenuipes extract exerted the mutagenic potential in Ames assay. Karyomegaly Since karyomegalic alterations have been known to be associated with carcinogenicity, our ﬁnding on the mutagenicity of P. tenuipes extract supports the possibility on the potential involvement of P. tenuipes in carcinogenicity at least partially. In conclusion, the subchronic oral exposure of P. tenuipes may induce kidney abnormality at the concentration higher than 500 mg/kg body weight, although further studies using other animal models are needed to identify the toxicity of P. tenuipes. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Dongchunghacho in Korea because of its appearance (Nam et al., 2001). This entomogenous fungus has long been widely used as Paecilomyces tenuipes, an entomogenous fungus on the health food ingredients and traditional nutritious medicines, espe- lepidopteran larvae, pupae, and adults, is called snow-ﬂake cially for allergic diseases, asthma, cancer and tuberculosis in Asian countries (Zhu et al., 1998a,b). In addition, the P. tenuipes has been known to contain novel ingredients that induce cellular differenti- Abbreviations: SD, Sprague Dawley; OECD, Organization for Economic Co- operation and Development; WBC, white blood cell; RBC, red blood cell; HGB, ation and inhibit cell growth in various malignant cell lines (Shim hemoglobin; HCT, hematocrit; PLT, platelet; MCV, mean corpuscular volume; MCH, et al., 2000, 2001) and to induce apoptosis in a human leukemic mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentra- cell line (Park et al., 2000). The P. tenuipes has also been reported tion; BUN, blood urea nitrogen; TC, total cholesterol; TP, total protein; TB, total to improve lipid proﬁles in rats fed a high fat diet (Koh and Choi, bilirubin; ALP, alkaline phosphatase; AST, aspartate transaminase; ALT, alanine 2003). And, Schmidt et al. (2003) demonstrated that the P. tenuipes transaminase; TG, triglyceride; CHL, Chinese hamster lung; FBS, fetal bovine serum; PCE, polychromatic erythrocytes. could play an important role in aging process through the modula- ⇑ Corresponding author at: Graduate School of Translational Medicine, Seoul tion of monoamine oxidase inhibitory activity and subsequent National University College of Medicine, 101 Daehak-ro, Jongno-gu, 110-744 Seoul, contribution to oxidative stress. Republic of Korea. Fax: +82 2 741 7620. Several reports on toxicity of Paecilomyces species were pub- E-mail address: [email protected] (B.-C. Kang). lished previously. Two-week oral treatment with complex powder 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.yrtph.2014.09.003 0273-2300/Ó 2014 Elsevier Inc. All rights reserved. 528 J.-H. Che et al. / Regulatory Toxicology and Pharmacology 70 (2014) 527–534 suspension of P. sinclairii and larvae of its host Bombyx mori did not 2.2. Acute oral toxicity study induce any toxicological effects in adult Sprague Dawley (SD) rats

(Kwack and Lee, 2009). The acute oral LD50 of this complex was The study was conducted in accordance with the Organization found to be greater than 5 g/kg of body weight in rats and dogs for Economic Co-operation and Development (OECD) test guideline (Kim et al., 1996). And, the results of a battery of in vitro and 420 (OECD, 2001). After quarantine and acclimatization, healthy in vivo genotoxicity tests indicated that this complex does not pos- rats of either sex were divided into two groups of 10 animals each sess any mutagenic or genotoxic potential (Ahn et al., 2004a). (5 males and 5 females). The rats were fasted for 16 h prior to con- However, there has been controversy over whether Paecilomyces ducting the experiment but had free access to water. The rats were species is beneficial or toxic to kidney function. Jeong et al. orally administered with the P. tenuipes extract at a dose of 0 or (2012) revealed that P. sinclairii exposed orally induced kidney cell 2000 mg/kg of body weight. On the day of dosing, all rats were damage and filtration dysfunction. The complex of P. sinclairii fruit- observed for mortality and signs of toxicity for several hours after ing body and silkworm larvae was also known to induce tubular dosing and once daily thereafter for 14 days. Body weights were cell abnormalities, including tubular edemas and tubular destruc- recorded on the day of treatment and on test day 1, 7 and 14. At tion, in kidney (Ahn et al., 2004b). In contrast, there was another the end of the study, all surviving rats were anesthetized with report on beneficial effect of Paecilomyces species on kidney isoflurane, and blood was collected via the posterior vena cava functions. Zhu et al. (1998b) identified that Cordyceps mushrooms from anesthetized animals. supported kidney functions through an increase in 17-hydroxy- corticosteroid and 17-ketosteroid levels. For these reasons, further studies are required to elucidate the effect of Paecilomyces species 2.3. Subchronic oral toxicity study on kidney functions. Especially, among Paecilomyces species, the toxicity of the P. tenuipes and underlying mechanism remains The study was performed according to the OECD test guideline unclear yet. Therefore, we performed the genotoxicity, acute and 408 (OECD, 1997a). Groups of 10 rats of each sex were orally trea- subchronic oral toxicity to investigate the potential hazards and ted with 0, 500, 1000, 2000 mg/kg P. tenuipes extract daily for safety concerns associated with the P. tenuipes. 13 weeks. The rats were observed daily for clinical signs and mortality, and body weights were measured every week during the 2. Materials and methods study period. At the last week of treatment, urinalysis of 10 rats (5 males and 5 females) per group was performed using fresh urine 2.1. Test substance and animals to determine pH, specific gravity, leukocyte, nitrite, protein, ketone body, urobilinogen, bilirubin, glucose, and occult blood using urine The P. tenuipes extract was kindly provided by Korea Food analyzer (Miditron Junior II, Roche, Mannheim, Germany). At the Research Institute (Seongnam, Korea). In brief, fruiting bodies of end of the study, all rats were anesthetized with isoflurane, and P. tenuipes were collected, dried and homogenized into powder- blood was collected from posterior vena cava. form. 100 g of power was macerated with 1 liter of water at Blood samples collected in an EDTA blood collection tube for 110 °C for 10 h. The suspension was filtered through filter paper hematology analysis were assayed using an automatic hematology for 5 h. After centrifugation at 6000 rpm for 20 min, the superna- analyzer ADVIA 2120i (Siemens Diagnostics, Tarrytown, NY, USA) tant of the extract was freeze–dried. It was resuspended with dis- for the following parameters: total white blood cell (WBC), red tilled water. SD rats and ICR mice were obtained from Orient bio blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), platelet (Seongnam, Korea) and housed in an environmentally-conditioned (PLT), mean corpuscular volume (MCV), mean corpuscular hemo- room (22 ± 2 °C, 40–60% humidity, and 12 h light cycle). The ani- globin (MCH), mean corpuscular hemoglobin concentration mals were allowed free access to rodent diet (Purina, Seoul, Korea) (MCHC) and differential WBC. and tap water. All animal experiments were approved by the Insti- In serum biochemistry analysis, serum was analyzed using an tutional Animal Care and Use Committee of the Biomedical automatic chemistry analyzer 7070 (Hitachi, Tokyo, Japan) for Research Institute at Seoul National University Hospital. And, this blood urea nitrogen (BUN), creatinine, total cholesterol (TC), total study was performed in compliance with the Good Laboratory protein (TP), albumin, total bilirubin (TB), alkaline phosphatase Practices for toxicity test guidance issued by the Korea Food and (ALP), aspartate transaminase (AST), alanine transaminase (ALT), Drug Administration (KFDA, 2005). triglyceride (TG), glucose, K, Cl, Ca, and P.

Fig. 1. Growth curves for male and female SD rats orally administered with P. tenuipes for 13 weeks. Data expressed as means ± SD. J.-H. Che et al. / Regulatory Toxicology and Pharmacology 70 (2014) 527–534 529

Fig. 2. Food consumption changes for male and female SD rats orally administered with P. tenuipes for 13 weeks. Data expressed as means ± SD.

At necropsy, heart, liver, lung, spleen, thymus, kidney, adrenal five Salmonella typhimurium strains TA98, 100, 102, 1535, and gland, testis, ovary, brain and pituitary gland were removed, 1537, which were provided from Ministry of Food and Drug Safety weighed and fixed in 10% neutral formalin, except for testis and (Osong, Korea). Strains were treated with the P. tenuipes extract in epididymis fixed in Bouin’s solution and eyes with Harderian the absence or presence of exogenous metabolic activation (S9 glands in Davidson solution. Nasal cavity, spinal cords with bones, mix) in the dark at 37 °C for 48 h. 2-nitrofluorene, sodium azide, sternum, and femora were treated with the decalcification solution mitomycin C, 9-aminoacridine, and 2-aminoanthracene (Sigma– for up to 3 weeks. Tissue slices of all tissues were embedded in par- Aldrich, St. Louis, MO, USA) were used as positive controls. The affin, sectioned and stained with hematoxylin and eosin. results were determined to be positive if the number of revertant colonies on the test plates doubled in comparison to negative con- 2.4. Genotoxicity study trol, and if there was an observed dose-related response. The in vitro chromosomal aberration test in Chinese hamster The bacterial reverse mutation assay (Ames test) was conducted lung (CHL) fibroblast cells was conducted in accordance with the in accordance with the OECD guideline 471 (OECD, 1997b). The OECD guideline 472 (OECD, 1997c). CHL fibroblast cells were potential mutagenicity of the P. tenuipes extract was examined in seeded at a density of 1 Â 105 cells/ml in Minimum Essential

Table 1 Hematological data for male and female SD rats orally administered with P. tenuipes for 13 weeks.

Item Dose of P. tenuipes (mg/kg) 0 500 1000 2000 Males WBC (103/mm3) 9.0 ± 1.7 8.5 ± 2.0 7.4 ± 2.7 8.1 ± 2.0 RBC (106/mm3) 7.7 ± 0.3 7.8 ± 0.3 8.0 ± 0.4 7.6 ± 0.4 HGB (g/dl) 15.3 ± 0.4 15.3 ± 0.5 16.1 ± 0.8 15.1 ± 0.6 HCT (%) 37.8 ± 1.2 37.8 ± 1.2 39.1 ± 1.1 37.0 ± 1.7 PLT (103/mm3) 905.5 ± 150.9 855.4 ± 157.4 968.8 ± 164.7 919.0 ± 149.7 MCV (ﬂ) 49.4 ± 1.4 48.6 ± 1.1 49.0 ± 1.7 49.0 ± 1.4 MCH (pg) 20.0 ± 0.5 19.6 ± 0.6 20.1 ± 1.2 19.9 ± 0.6 MCHC (g/dl) 40.6 ± 0.5 40.5 ± 0.8 41.1 ± 1.6 40.8 ± 0.6 Neutrophils (%) 7.3 ± 5.0 8.6 ± 5.4 11.8 ± 5.4 19.1 ± 8.5* Eosinophils (%) 1.4 ± 1.2 1.0 ± 1.1 1.0 ± 0.7 1.6 ± 1.4 Basophils (%) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Lymphocytes (%) 86.1 ± 6.2 87.8 ± 4.8 83.3 ± 4.9 74.0 ± 11.1* Monocytes (%) 5.4 ± 4.7 2.7 ± 2.1 3.9 ± 2.1 5.3 ± 3.4 Reticulocyte (%) 0.8 ± 0.4 0.7 ± 0.3 0.6 ± 0.4 0.5 ± 0.1 Females WBC (103/mm3) 6.6 ± 1.7 6.1 ± 0.9 5.7 ± 2.2 6.1 ± 1.5 RBC (106/mm3) 6.9 ± 0.3 6.8 ± 0.3 7.1 ± 0.3 7.0 ± 0.3 HGB (g/dl) 15.0 ± 0.5 14.6 ± 0.5 15.0 ± 0.5 14.7 ± 0.4 HCT (%) 36.5 ± 1.3 35.6 ± 1.1 36.8 ± 1.3 35.8 ± 1.4 PLT (103/mm3) 862.8 ± 141.2 880.9 ± 106.2 881.2 ± 130.8 906.9 ± 88.1 MCV (ﬂ) 52.9 ± 0.7 52.4 ± 2.6 52.1 ± 1.9 51.2 ± 1.6 MCH (pg) 21.8 ± 0.4 21.5 ± 1.0 21.2 ± 0.8 21.0 ± 0.7 MCHC (g/dl) 41.2 ± 0.5 41.1 ± 0.4 40.7 ± 0.3 41.1 ± 0.8 Neutrophils (%) 10.1 ± 8.6 8.6 ± 9.0 6.3 ± 3.9 6.2 ± 5.7 Eosinophils (%) 1.1 ± 1.3 1.0 ± 1.1 0.6 ± 0.7 1.0 ± 1.0 Basophils (%) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Lymphocytes (%) 87.6 ± 9.7 89.2 ± 9.5 91.3 ± 6.3 91.2 ± 7.3 Monocytes (%) 1.2 ± 1.0 1.4 ± 1.3 1.8 ± 2.5 1.6 ± 1.6 Reticulocyte (%) 0.6 ± 0.1 0.7 ± 0.2 0.6 ± 0.1 0.5 ± 0.1

* Significantly different from Control group (p < 0.05). 530 J.-H. Che et al. / Regulatory Toxicology and Pharmacology 70 (2014) 527–534 medium (GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal treated with the P. tenuipes extract (p<0.05) (Table 1). However, bovine serum (FBS, GIBCO) in 25 cm2 flask and incubated in a no significant treatment-related change in the other hematological

CO2 incubator (5% CO2,37°C, high humidity) for 24 h. After the parameters was noted in any of the P. tenuipes groups, compared to treatment with the P. tenuipes extract in the presence or absence the control group after the treatment for 13 weeks in male and of exogenous metabolic activation (S9 mix) for 6 h or 24 h, the cells female rats. On serum biochemical analysis, there were some were washed and incubated in complete medium for 18 h. changes among groups, however, these were not considered Mitomycin C and cyclophosphamide (Sigma–Aldrich) were used adverse or specifically related to the treatment of the P. tenuipes as positive controls. After colcemid (0.2 lg/ml, GIBCO) was added extract because the changes were sporadic, and were of small mag- for 2 h, the cells were treated with hypotonic solution, fixed in nitude (Table 2). 3:1 methanol/glacial acetic acid, and stained with 4% Giemsa. The in vivo bone marrow micronucleus test in mice was con- 3.2.3. Organ weights and histopathological changes ducted in compliance with OECD guideline 474 (OECD, 1997d). The data from final body and organ weights are summarized in Male ICR mice were orally gavaged with 0, 500, 1000, 2000 mg/ Table 3. There was no treatment-related change in mean absolute kg P. tenuipes extract dissolved in 1% methylcellulose daily for and relative organ weights following the P. tenuipes extract treat- 4 d. Mitomycin C used as a positive control was administered via ment in male and female rats. a single intraperitoneal injection at a dose of 2 mg/kg of body Histopathological findings associated with the treatment of the weight. After the mice were sacrificed at 24 h after the last dose P. tenuipes extract were elucidated in kidney tissues of male and of the P. tenuipes extract, femoral bone marrow cells were isolated female rats at the end of the administration period of 13 weeks from each mouse. The cells were centrifuged, smeared onto slides, (Fig. 3). In the outer medulla, kidney cell karyomegaly, that is dried, and fixed in methanol. The slides were stained with 5% abnormal nuclear enlargement, was observed at all of the oral Giemsa and evaluated for at least 2000 polychromatic erythrocytes treatment doses of the P. tenuipes extract fed to the rats with sig- (PCE) for the presence of micronuclei per animal. And, the ratio of nificant increases in both incidence and severity although there polychromatic to all erythrocytes (normochromatic erythrocytes were no clear dose-dependence in the severity of karyomegaly (NCE) and polychromatic erythrocytes) was also determined. (Table 4). It was interesting to note that the male rats were more severely affected than the female rats. In the other organs exam- 2.5. Statistical analysis ined, spontaneous lesions on histopathology were observed in both the untreated controls and the P. tenuipes-treated groups, but with- The data were expressed as means ± SD. All data were analyzed out statistical difference in the incidence and/or severity among by a one-way ANOVA using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). If the variance was significant (p < 0.05), the data were analyzed by the multiple comparison procedure of Tukey Table 2 test. Incidences for histopathological lesions were compared using Serum biochemistry data for male and female SD rats orally administered with P. Chi-square test. tenuipes for 13 weeks.

Item Dose of P. tenuipes (mg/kg) 3. Results 0 500 1000 2000 Males 3.1. Acute oral toxicity study BUN (mg/dL) 12.1 ± 1.3 9.3 ± 1.9* 10.6 ± 1.6 9.7 ± 1.7* TC (mg/dL) 59.6 ± 15.2 47.6 ± 9.1 52.3 ± 8.1 45.0 ± 6.9* At the acute trial, the single oral administration of the TP (g/dL) 5.1 ± 0.2 5.0 ± 0.3 5.2 ± 0.3 5.1 ± 0.2 * P. tenuipes extract at 2000 mg/kg of body weight caused no abnor- Albumin (g/dL) 1.9 ± 0.1 2.0 ± 0.1 2.1 ± 0.1 2.0 ± 0.1 TB (mg/dL) 0.03 ± 0.05 0.00 ± 0.00 0.01 ± 0.03 0.00 ± 0.00 mal clinical sign during the experimental period. Likewise, there ALP (IU/L) 96.4 ± 22.5 75.0 ± 9.9* 87.0 ± 13.3 97.4 ± 11.1 was no significant change in body weight in the P. tenuipes extract AST (IU/L) 70.4 ± 14.3 83.2 ± 11.9 78.9 ± 14.6 83.1 ± 20.8 treatment groups in comparison to the control group during the ALT (IU/L) 28.8 ± 4.3 28.7 ± 6.6 27.6 ± 4.4 33.4 ± 6.1 study period. At necropsy after the 14-day observation period, no Creatinine (mg/dL) 0.53 ± 0.05 0.57 ± 0.07 0.57 ± 0.05 0.56 ± 0.05 macroscopic lesion was observed in any animals. Thus, the acute TG (mg/dL) 88.5 ± 40.4 72.8 ± 33.7 85.6 ± 26.3 62.7 ± 29.0 Glucose (mg/L) 117.8 ± 13.3 125.7 ± 12.1 131.9 ± 16.8 121.9 ± 9.6 oral LD50 value of the P. tenuipes extract was determined to be K (mmol/L) 4.4 ± 0.3 4.5 ± 0.2 4.6 ± 0.2 4.4 ± 0.3 greater than 2000 mg/kg of body weight. Cl (mmol/L) 104.7 ± 2.1 105.9 ± 1.6 105.6 ± 0.8 106.4 ± 1.2 Ca (mg/dL) 8.4 ± 0.2 8.3 ± 0.2 8.5 ± 0.3 8.5 ± 0.3 P (mg/dL) 5.9 ± 0.5 6.0 ± 0.5 5.9 ± 0.7 5.7 ± 0.6 3.2. Subchronic toxicity study * Na (mmol/L) 147.2 ± 0.8 146.1 ± 0.8 145.6 ± 1.1 146.3 ± 1.4 Females 3.2.1. Changes in body weight and daily feed intake BUN (mg/dL) 12.8 ± 1.9 12.6 ± 1.6 12.0 ± 1.9 11.1 ± 2.4 To identify the effects of subchronic exposure of the P. tenuipes TC (mg/dL) 73.0 ± 10.7 64.2 ± 10.7 74.7 ± 15.5 61.9 ± 7.8 extract, the rats were gavaged with 0, 500, 1000, 2000 mg/kg P. TP (g/dL) 5.7 ± 0.3 5.7 ± 0.3 5.4 ± 0.5 5.2 ± 0.2* tenuipes daily for 13 weeks. No significant difference of body Albumin (g/dL) 2.5 ± 0.2 2.5 ± 0.2 2.4 ± 0.2 2.3 ± 0.2 weight was observed between the control and the P. tenuipes TB (mg/dL) 0.07 ± 0.05 0.04 ± 0.05 0.04 ± 0.05 0.01 ± 0.03 ALP (IU/L) 45.0 ± 7.5 51.8 ± 11.2 40.4 ± 12.4 47.2 ± 9.1 extract groups throughout the feeding period (Fig. 1). And, there AST (IU/L) 81.4 ± 13.8 87.2 ± 21.5 71.2 ± 9.3 65.1 ± 19.5 was no difference of mean daily food consumption among groups ALT (IU/L) 23.7 ± 5.2 30.9 ± 12.3 23.1 ± 5.8 23.6 ± 3.8 throughout whole experimental period (Fig. 2). Creatinine (mg/dL) 0.61 ± 0.07 0.62 ± 0.07 0.59 ± 0.06 0.60 ± 0.05 TG (mg/dL) 23.4 ± 14.0 23.9 ± 9.0 33.4 ± 34.3 17.3 ± 5.1 Glucose (mg/L) 120.6 ± 10.3 122.7 ± 9.6 119.8 ± 10.7 127.4 ± 8.5 3.2.2. Urinalysis, hematology and clinical chemistry K (mmol/L) 4.3 ± 0.2 4.4 ± 0.2 4.4 ± 0.2 4.2 ± 0.3 Urinalysis revealed that all observed changes were not consid- Cl (mmol/L) 108.3 ± 2.8 110.2 ± 2.1 106.1 ± 3.6 106.6 ± 2.6 * ered any treatment-related adverse effects following the P. tenuipes Ca (mg/dL) 9.0 ± 0.3 9.0 ± 0.4 8.6 ± 0.4 8.4 ± 0.3 extract administration (Data not shown). On hematological analy- P (mg/dL) 5.0 ± 0.9 5.0 ± 0.5 4.4 ± 0.7 4.3 ± 0.8 Na (mmol/L) 148.8 ± 2.3 148.7 ± 2.0 143.7 ± 3.9* 142.5 ± 1.3* sis, WBC differential showed an increase in the percentage of neutrophils and a decrease in the percentage of lymphocytes in males * Significantly different from Control group (p < 0.05). J.-H. Che et al. / Regulatory Toxicology and Pharmacology 70 (2014) 527–534 531

Table 3 Final body and organ weights for male and female SD rats orally administered with P. tenuipes for 13 weeks.

Item Dose of P. tenuipes (mg/kg) 0 500 1000 2000 Males Body weight (g) 568.4 ± 57.5 573.2 ± 44.4 528.5 ± 53.4 549.3 ± 28.9 Liver (g) 15.50 ± 1.55 14.95 ± 1.78 13.74 ± 1.48 14.18 ± 1.55 (%BW) 2.73 ± 0.12 2.61 ± 0.23 2.60 ± 0.18 2.58 ± 0.25 Spleen (g) 0.99 ± 0.18 0.86 ± 0.07 0.74 ± 0.08* 0.86 ± 0.08 (%BW) 0.17 ± 0.02 0.15 ± 0.01 0.14 ± 0.02* 0.16 ± 0.02 Kidney (g) 1.94 ± 0.17 1.82 ± 0.13 1.64 ± 0.20* 1.80 ± 0.18 (%BW) 0.34 ± 0.03 0.32 ± 0.01 0.31 ± 0.04 0.33 ± 0.04 Adrenal gl. (g) 0.031 ± 0.007 0.029 ± 0.004 0.028 ± 0.005 0.029 ± 0.005 (%BW) 0.006 ± 0.001 0.005 ± 0.001 0.005 ± 0.001 0.005 ± 0.001 Testis (g) 1.71 ± 0.10 1.68 ± 0.13 1.70 ± 0.09 1.70 ± 0.10 (%BW) 0.30 ± 0.02 0.29 ± 0.03 0.33 ± 0.04 0.31 ± 0.03 Thymus (g) 0.31 ± 0.09 0.32 ± 0.08 0.29 ± 0.09 0.34 ± 0.06 (%BW) 0.05 ± 0.01 0.06 ± 0.01 0.06 ± 0.02 0.06 ± 0.01 Heart (g) 1.63 ± 0.18 1.69 ± 0.14 1.50 ± 0.09 1.58 ± 0.10 (%BW) 0.29 ± 0.02 0.29 ± 0.02 0.29 ± 0.03 0.29 ± 0.02 Lung (g) 1.73 ± 0.22 1.65 ± 0.09 1.57 ± 0.20 1.67 ± 0.16 (%BW) 0.31 ± 0.06 0.29 ± 0.02 0.30 ± 0.05 0.31 ± 0.04 Brain (g) 2.23 ± 0.07 2.19 ± 0.09 2.13 ± 0.05 2.20 ± 0.14 (%BW) 0.40 ± 0.04 0.38 ± 0.02 0.41 ± 0.04 0.40 ± 0.04 Pitui gl. (g) 0.015 ± 0.003 0.014 ± 0.001 0.014 ± 0.002 0.016 ± 0.004 (%BW) 0.003 ± 0.001 0.002 ± 0.000 0.003 ± 0.000 0.003 ± 0.001 Females Body weight (g) 272.1 ± 24.4 263.6 ± 28.1 278.3 ± 27.1 279.5 ± 19.3 Liver (g) 7.05 ± 0.58 7.03 ± 0.70 7.05 ± 0.71 7.33 ± 0.65 (%BW) 2.60 ± 0.15 2.67 ± 0.14 2.54 ± 0.19 2.62 ± 0.14 Spleen (g) 0.50 ± 0.06 0.52 ± 0.04 0.51 ± 0.05 0.55 ± 0.08 (%BW) 0.18 ± 0.01 0.20 ± 0.02 0.18 ± 0.02 0.19 ± 0.02 Kidney (g) 0.92 ± 0.12 0.86 ± 0.07 0.88 ± 0.05 0.89 ± 0.08 (%BW) 0.34 ± 0.03 0.33 ± 0.03 0.32 ± 0.03 0.32 ± 0.02 Adrenal gl. (g) 0.031 ± 0.004 0.033 ± 0.005 0.031 ± 0.005 0.030 ± 0.003 (%BW) 0.012 ± 0.001 0.013 ± 0.002 0.011 ± 0.003 0.011 ± 0.001 Ovary (g) 0.051 ± 0.006 0.055 ± 0.011 0.051 ± 0.011 0.056 ± 0.012 (%BW) 0.019 ± 0.002 0.021 ± 0.004 0.019 ± 0.005 0.020 ± 0.004 Thymus (g) 0.24 ± 0.05 0.26 ± 0.05 0.30 ± 0.04 0.28 ± 0.06 (%BW) 0.09 ± 0.01 0.10 ± 0.02 0.11 ± 0.02 0.10 ± 0.02 Heart (g) 0.90 ± 0.08 0.91 ± 0.08 0.92 ± 0.05 0.94 ± 0.06 (%BW) 0.33 ± 0.02 0.35 ± 0.03 0.33 ± 0.02 0.34 ± 0.03 Lung (g) 1.14 ± 0.09 1.14 ± 0.11 1.15 ± 0.09 1.16 ± 0.07 (%BW) 0.42 ± 0.06 0.44 ± 0.04 0.41 ± 0.05 0.41 ± 0.02 Brain (g) 1.91 ± 0.06 1.96 ± 0.11 1.91 ± 0.07 1.93 ± 0.09 (%BW) 0.71 ± 0.06 0.75 ± 0.08 0.69 ± 0.08 0.69 ± 0.05 Pitui gl. (g) 0.016 ± 0.002 0.018 ± 0.003 0.017 ± 0.002 0.017 ± 0.003 (%BW) 0.006 ± 0.001 0.007 ± 0.002 0.006 ± 0.001 0.006 ± 0.001

* Signiﬁcantly different from Control group (p < 0.05).

groups (Data not shown), indicating that these changes were not with or without S9 mix, whereas no evidence of significant considered toxicologically relevant. increase in the incidence of chromosomal aberrations was observed at any of the concentration tested in CHL cells treated 3.3. Genotoxicity with the P. tenuipes extract in the presence or absence of S9 mix (Table 6). 3.3.1. Bacterial reverse mutation assay (Ames test) The positive controls showed increase in the number of revertant colonies with dose-dependent relationship, indicating the 3.3.3. In vivo bone marrow micronucleus assay validity of the study (Table 5). The P. tenuipes extract significantly Since micronuclei are indirect indicators of structural chromo- produced His+ mutants at the highest concentration 5000 lg/plate somal aberrations (Akyil and Konuk, 2014), in vivo micronucleus in S. typhimurium TA102 and 1535 without S9 mix. In the presence assay as a more reliable method was conducted to assess the geno- of S9 mix, it also significantly produced His+ mutants in TA98, 100 toxic effect of the P. tenuipes, and found that significant increases in and 102 at 5000 lg/plate of the P. tenuipes extract. These increases the micronucleated polychromatic erythrocytes (MNPCE) and in His+ mutants were dose-dependent although they were less decreases in PCE/(PCE + NCE) ratio were observed in mice treated than 2-fold in comparison to the control group. with the positive control, mitomycin C (Table 7). In contrast, no significant decreases in PCE/(PCE + NCE) ratio was found in mice 3.3.2. Chromosomal aberration assay treated with the P. tenuipes extract at any doses tested. And, P. ten- The different chromosomal aberrations, including breaks, frag- uipes extract did not cause any statistically significant increase in ments, exchanges, and other multiple damages, were analyzed. MNPCE at concentrations of 500, 1000 and 2000 mg/kg of body Statistically significant increases in the incidence of structural weight, suggesting that the P. tenuipes is not clastogenic after chromosome aberrations were noted in the positive control groups in vivo exposure. 532 J.-H. Che et al. / Regulatory Toxicology and Pharmacology 70 (2014) 527–534

Fig. 3. Histopathological changes in the kidneys of SD rats orally administered with P. tenuipes for 13 weeks. Representative images of kidney tissue stained with H and E following P. tenuipes. (A) Normal in outer stripe of outer medulla (Male, Vehicle control, X40). (B) Slight karyomegalic alteration (Female, P. tenuipes 2000 mg/kg, X100). (C) Marked karyomegalic alteration (Male, P. tenuipes 2000 mg/kg, X100). (D) Enlarged nucleus (Male, P. tenuipes 2000 mg/kg, X400).

Table 4 hematological and serum biochemical data. Although the level of Incidence and severity of karyomegalic alteration in kidney of male and female SD neutrophil was significantly increased at a dose-dependent man- rats orally administered with P. tenuipes for 13 weeks. ner in the P. tenuipes extract-treated male rats, histopathological Item Dose of P. tenuipes (mg/kg) examination indicated that no acute inflammatory lesion was Males (n = 10/group) Females (n = 10/group) observed following the treatment of the P. tenuipes extract. In contrast, the P. tenuipes extract exerted the karyomegalic alteration in 0 500 1000 2000 0 500 1000 2000 the outer medulla of kidney despite no significant changes of Karyomegalic alteration serum biochemistry markers. Renal function is routinely moni- Normal 10 0 0 0 10 0 0 0 Slight 0 0 0 0 0 2 1 5 tored by serum biochemistry markers such as serum creatinine Moderate 0 0 0 0 0 8 9 5 and BUN. However, these serum biochemical indicators often Marked 0 10 10 10 0 0 0 0 change only when a significant level of kidney function is damaged (Perazella, 2009), which helps explain the difference of our results between histopathological and biochemical analysis for early 4. Discussion detection and diagnosis of kidney injury. The P. tenuipes has been known to induce cellular differentia- P. tenuipes, an entomogenous fungus on the larvae of lepidop- tion and inhibit cell growth in various malignant cell lines, and tera, is widely used in traditional nutritious medicine in the Orient to induce apoptosis in human breast cancer and leukemic cell lines, (Lee et al., 2006). In spite of lots of reports on the beneficial activ- indicating that the P. tenuipes could contain potential oncostatic ities of Paecilomyces species, there have also been several studies on and antitumor components (Park et al., 2000; Shim et al., 2000, the toxicities of Paecilomyces species, especially P. sinclairii, includ- 2001; Nam et al., 2001; Chung et al., 2003). Nevertheless, karyo- ing acute oral toxicity studies in dogs, subchronic oral toxicity megaly induced in the outer medulla by the P. tenuipes extract in study in rats and a series of genotoxicity studies (Ahn et al., our study has been known as a typical histological feature in kid- 2003, 2004a,b; Jeong et al., 2012, 2013). Among Paecilomyces spe- ney before the development of tumors in a spontaneous renal cell cies, the P. tenuipes-contained processed goods have also been tumor of Long-Evans Cinnamon rats (Kitaura et al., 1999). Also, developed in many countries, particularly South Korea (Nam renal tubular cell karyomegaly in the outer medulla was often et al., 2011). In order to identify the toxicity of the P. tenuipes,we reported in relation with the carcinogenicity in rodents, irrespec- performed the genotoxicity, acute and subchronic oral toxicity tive of the genotoxic potential (US NTP, 1996; Hard et al., 2000; studies in rats. Greim and Reuter, 2001; Lock and Reed, 2006). Ochratoxin A

In the present study, the acute oral LD50 in rats for the P. tenu- (OTA), a mycotoxin mostly produced by Aspergillus ochraceus and ipes extract has been estimated to be greater than 2000 mg/kg of Penicillium verrucosum, has been known as a renal carcinogen body weight. In the subchronic toxicity study, the rats tested and (Boesch-Saadatmandi et al., 2008). Boorman et al. (1992) reported compared with controls did not show any treatment-related differ- that karyomegaly has been induced in the proximal tubular epithe- ences in terms of body weight and food consumption. And, our cur- lial cells of the outer stripe of the outer medulla in rats following rent results also indicated that the oral dosing of rats with the P. the administration of OTA. Importantly, our results showed that tenuipes extract showed no treatment-related changes of the P. tenuipes extract was found to have mutagenic potential at J.-H. Che et al. / Regulatory Toxicology and Pharmacology 70 (2014) 527–534 533

Table 5 Results of S. typhimurium reversion assay with P. tenuipes extract.

S9 Chemical Dose (lg/plate) His+ revertant colonys/plate TA98 TA100 TA102 TA1535 TA1537

À Distilled watera – 23 ± 4 210 ± 6 363 ± 8 16 ± 1 99 ± 10 2-nitroﬂuoreneb 10 407 ± 21* –––– Sodium azideb 5 – 2668 ± 241* ––– Mitomycin Cb 0.5 – – 1498 ± 175* –– Sodium azideb 0.5 – – – 368 ± 11* – 9-aminoacridineb 80 – – – – 2540 ± 73* P. tenuipes 312.5 19 ± 1 152 ± 6 453 ± 40 17 ± 2 98 ± 3 625 20 ± 2 150 ± 3 423 ± 4 17 ± 3 104 ± 4 1250 25 ± 1 163 ± 5 446 ± 15 18 ± 1 108 ± 4 2500 26 ± 2 179 ± 7 598 ± 3* 22 ± 1 109 ± 3 5000 26 ± 2 252 ± 4 633 ± 22* 30 ± 2* 145 ± 5 + Distilled water – 35 ± 3 235 ± 12 463 ± 26 17 ± 1 97 ± 5 2-aminoanthraceneb 2 257 ± 17* 451 ± 32* ––– 5 – – 901 ± 39* 349 ± 27* 332 ± 32* P. tenuipes 312.5 39 ± 1 258 ± 7 490 ± 20 18 ± 2 94 ± 5 625 40 ± 2 254 ± 12 493 ± 13 18 ± 0 94 ± 4 1250 39 ± 4 283 ± 8 493 ± 17 18 ± 2 93 ± 5 2500 40 ± 4 331 ± 22* 585 ± 15* 18 ± 2 109 ± 4 5000 64 ± 5* 354 ± 23* 586 ± 14* 26 ± 1 115 ± 7

a Negative control. b Positive control. * Signiﬁcantly different from negative control group (p < 0.05).

Table 6 Results of chromosomal aberration induced by P. tenuipes extract.

Substance Dose (lg/ml) Number of cells scored No. of cells with aberrations –S9 +S9 6 h 24 h 6 h MEMa – 200 3.5 ± 0.7 3.5 ± 2.1 4.0 ± 2.8 Mitomycin Cb 0.1 200 30.0 ± 17.0* 57.0 ± 9.9* – Cyclophosphoamideb 5 200 – – 84.0 ± 2.8* Distilled water – 200 4.0 ± 0.0 1.5 ± 0.7 6.0 ± 5.7 P. tenuipes 625 200 1.5 ± 0.7 1.5 ± 0.7 1.0 ± 0.0 1250 200 2.0 ± 1.4 1.0 ± 0.0 1.5 ± 2.1 2500 200 5.5 ± 0.7 1.0 ± 1.4 3.0 ± 1.4

a Minimum essential medium (negative control). b Positive control. * Signiﬁcantly different from negative control group (p < 0.05).

Table 7 Micronucleated polychromatic erythrocytes (MNPCEs) in mice bone marrow following treatment with P. tenuipes extract.

Substance Dosage (mg/kg BW) Number of mice MNPCEc PCE/(PCE + NCE)d Distilled watera 0.0 5 0.2 ± 0.5 46.9 ± 6.8 P. tenuipes 500 5 0.6 ± 0.6 41.4 ± 2.0 1000 5 0.2 ± 0.5 38.2 ± 2.8 2000 5 0.2 ± 0.5 39.1 ± 4.2 Mitomycin Cb 2 5 52.0 ± 12.0* 24.6 ± 2.3*

a Negative control. b Positive control. c Polychromatic erythrocyte with micronuclei was calculated from 2000 polychromatic erythrocytes (%). d The ratio of polychromatic erythrocytes to all erythrocytes (polychromatic + normochromatic) (%). * Signiﬁcantly different from negative control group (p < 0.05). the highest concentration tested as evidenced by Ames assay. estrogen activity (Muller et al., 2002; Antus et al., 2003; Wei Therefore, our histopathological ﬁnding on kidney cell karyomega- et al., 2005), supporting our results on the difference of susceptibil- ly induced by the P. tenuipes may provide the evidence on the ity to kidney injury between male and female rats. potential role of the P. tenuipes in the development of tumor The aforementioned toxicity studies were conducted to investi- although further study on carcinogenicity is needed. Noteworthy, gate the safety concern of the P. tenuipes and found that the P. ten- kidney changes caused by the P. tenuipes extract were more severe uipes extract exposed orally could induce kidney cell damage, in male animals than females in the histopathological lesions of including karyomegalic alteration in the outer medulla. Previous renal tubule cells in agreement with recent result on the P. sinclairii study reported that the administration of lysinoalanine, the amino (Jeong et al., 2013). Female rodents have been reported to resist acid known to be formed in proteins subjected to high pH at ele- ischemic acute renal failure or glomerulosclerosis through the vated temperature, resulted in karyomegalic changes in kidney 534 J.-H. Che et al. / Regulatory Toxicology and Pharmacology 70 (2014) 527–534

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