39 Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 3 (supplement): 39–46, 2018, ISSN 2543-8832 DOI: 10.24917/25438832.3supp.5 Vendula Kuchařová1*, Ondřej Daněk1, Miša Škorič2, Ivana Veselá1, Jaroslava Tomenendálová1 1 Department of Physiology, University of Veterinary and Pharmaceutical Sciences, Brno, Czechia, *kucharova.vendula@gmail.com 2Department of Pathological Morphology and Parasitology, University of Veterinary and Pharmaceutical Sciences Brno, Czechia Organ toxicity of diethylnitrosamine and capsaicin in mice – in vivo study Introduction Application of diethylnitrosamine (DEN) is commonly used in chemically induced animal models for hepatotoxicity and hepatocarcinogenesis. DEN belongs to a wide range of nitrosamines, substances well known for their general toxic properties. Al- ready in the 1930s, Freund (1937) described the hepatotoxic e�ect of one of these substances, dimethylnitrosamine. Application of DEN in di�erent doses and regimes was shown to induce histopathological changes in liver and kidneys a�ecting the bi- ochemical parameters of hepatic injury (Rezaie et al., 2013; Paula-Santos et al., 2014; Cho et al., 2016; Shaker et al., 2016). Capsaicin (CAP) is the main pungent principle in Capsicum fruit, whose physi- cal and chemical properties and biological e�ects have been already described and summarised more than 30 years ago (Monsereenusorn et al., 1982). In more recent articles, various biological activities are described, such as thermogenic and weight re- ducing, analgesic, antioxidant, anti-in�ammatory, hypocholesterolemic and hypolipi- demic, anti-diabetic, anti-ulcer, anti-apoptotic, and anti-cancer (Basith et al., 2016; Srinivasan, 2016). However, the last-mentioned e�ect is still controversial. Some au- thors highlighted a possible dual e�ect of CAP in mutagenicity and tumorigenicity (Surh, Lee, 1996; Bode, Dong, 2011). Bley et al. (2012) highlight the source and meth- od of extraction of CAP and other capsaicinoid compounds which may have several impurities responsible for the potentially carcinogenicity e�ect. However, pure CAP is a rather chemopreventive substance according to their study (Bley et al., 2012). �e aim of our study was to assess the toxicity of DEN and CAP in mice liver and kidney and to evaluate the chemoprotective e�ect of CAP on DEN induced liver and kidney injury. 40 V en du la K uc ha řo vá , O nd ře j D an ěk , M iš a Š ko rič , I va na V es el á, J ar os la va T om en en dá lo vá Materials and methods Chemicals Diethylnitrosamine (DEN, Sigma-Aldrich) was dissolved in saline solution (0.9% NaCl). Capsaicin (CAP, Merck Millipore) was �rst dissolved in ethanol to a stock solution, and this was further dissolved in saline to form a �nal solution containing 0.1 CAP in 1 ml 1% ethanol solution (dosage 1.5 mg/kg body weight) or a �nal solu- tion containing 0.05 mg CAP in 1 ml 1% ethanol solution (dosage 0.75 mg/kg body weight). Animals and experimental conditions 51 female ICR mice at 4 weeks of age (13–16 g) were obtained from AnLab s.r.o. (Prague, Czech Republic). A�er 2 weeks of quarantine and acclimatisation, mice were randomly divided into �ve groups, housed in plastic cages with sawdust as bedding with a 12-h light/dark cycle, and temperature and humidity were monitored twice a day. Drinking water and a standard mouse diet (Biokron s.r.o., Blučina, Czech Repub- lic) was provided ad libitum throughout the experiment. All experimental procedures were conducted according to the Czech animal wel- fare protection legal guidelines and EU directives. Paper rolls were used to provide environmental enrichment. Experimental design and sample collection A total of 51 female ICR mice were randomly divided into �ve groups. Mice from the �rst group (control, n = 11) received the ethanol solution (1%) intraperitoneally (IP) in weeks 3, 5, 7, 9, and 11. Mice in the second group (CAP, n = 10) received CAP IP at a dose of 1.5 mg/kg body weight (bw) in weeks 3 and 5 and due to impaired tolerance the dose was halved in weeks 7, 9, and 11 (0.75 mg/kg bw). �ird group (DEN-CAP, n = 10) DEN IP at a dose of 25 mg/ kg bw was administered in week 2 and from week 3 until the end of the experiment as in the second group (CAP). Mice in the fourth group (CAP-DEN, n = 10) received CAP IP at a dose of 1.5 mg/kg bw in week 1 and a week a�er DEN IP at a dose 25 mg/kg bw. In weeks 3, 5, 7, 9, and 11, IP saline was ad- ministered. Mice in the last group (DEN, n = 10) received DEN IP at a dose of 25 mg/ kg bw in week 2 and in weeks 3, 5, 7, 9, and 11 saline IP. All procedures and applica- tions were performed between 8 and 12 hours, and 24 hours a�er the last application all mice were euthanized using ether overdose and decapitation. �roughout the experiment, animals were observed daily to assess their general health, and the body weight was measured weekly. A�er euthanization, blood was collected from each mouse and clotting serum was obtained. Complete necropsy was performed, and the livers and kidneys were weighed, examined macroscopically, �xed 41 O rgan toxicity of diethylnitrosam ine and capsaicin in m ice – in vivo study in 10% bu�ered formalin, and representative parts were embedded in para�n wax. Biochemical parameters assessment and histological evaluation Total serum protein (TP), albumin (Alb), and activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assessed by a clinical chemistry analyser, Abbott ARCHITECT c4000 (Abbott, Abbott Park, Illinois, U.S.A.). Tissue sections of 4 μm were processed and routinely stained with haematoxylins and eosin (H&E). Slides were observed under light microscopy by di�erent experi- enced histopathologists in a blind fashion and results were compared. Statistical analysis Numerical data was statistically processed by MedCalc for Windows, version 15.11.0 (MedCalc So�ware, Ostend, Belgium). ANOVA and Student t-tests were used. Results �ere was no statistically signi�cant di�erence in body and organ weight between control and experimental groups (data not shown). Levels of ALT, AST activity, total protein, and albumin concentration were not statistically di�erent among control and experimental groups (Tab. 1). Tab. 1. Results of biochemical parameters: TP – total serum protein, Alb – albumin, ALT – alanine ami- notransferase, AST – aspartate aminotransferase; values shown as mean ± SD Group TP [g/l] Alb [g/l] ALT [μkat/l] AST [μkat/l] Control 54.48 ±2.03 38.92 ±1.76 0.37 ±0.13 2.43 ±0.72 CAP 52.07 ±1.57 37.23 ±1.78 0.36 ±0.08 1.95 ±0.48 CAP-DEN 54.57 ±3.21 38.33 ±2.69 0.35 ±0.12 2.43 ±0.76 DEN-CAP 53.90 ±2.85 38.56 ±2.58 0.32 ±0.08 2.01 ±0.48 DEN 53.65 ±3.92 37.77 ±3.36 0.35 ±0.10 2.37 ±0.50 �e histopathological examination of the liver revealed multifocal lymphoplasma- cytic reaction in parenchyma in the DEN treated group (Fig. 1). CAP used as both pre- ventive and therapeutic agent caused a reduction in the number and extent of lesions. In the CAP group, mitotic �gures were found suggesting xenobiotic-induced hepatotoxicity or regenerative changes (Fig. 2). In the kidneys, DEN also revealed multifocal lymphop- lasmacytic reactions (Fig. 3) that have been mitigated by CAP. Moreover, histopatho- logical observation of the kidney in the DEN group has revealed granular dystrophy of the renal tubules (Fig. 4), which has not been presented in CAP treated mice. In the control group, histological changes were observed neither in the liver nor in the kidneys. V en du la K uc ha řo vá , O nd ře j D an ěk , M iš a Š ko rič , I va na V es el á, J ar os la va T om en en dá lo vá 42 Fig. 1. Lymphoplasmacytic reaction in liver parenchyma in DEN treated group. Original magni�cation: A 200×, B 400×; H&E stain. (Photo. Kuchařová et al.) Fig. 2. Mitotic �gures in liver parenchyma in CAP treated group. Original magni�cation: 400×; H&E stain. (Photo. Kuchařová et al.) Fig. 3. Lymphoplasmacytic reaction in the renal cortex in DEN treated group. Original magni�cation: A 100×, B 200×; H&E stain (Photo. Kuchařová et al.) 43 Discussion Intraperitoneal application of DEN to adult mice led to increased ALT and AST ac- tivities during the �rst 24 or 48 hours, suggesting a physiologic reaction to the acute toxicity of this chemical (Cho et al., 2016; Hanna et al., 2016; Shaker et al., 2016). When these parameters were assessed a�er a longer period a�er DEN application (20 or 40 weeks), the e�ect was not so prominent. Sun et al. (2012) described increased ALT activity a�er DEN-initiated and phenobarbital-promoted liver injury with no change in AST activity. However, Healy et al. (2016) did not observe any change in ALT activity a�er DEN application in female C57BL/6N mice. In our study, we did not �nd any DEN-induced changes in ALT and AST activity, which was probably caused due to the late sampling a�er DEN application (11 weeks a�er DEN administration). Capsaicin alone does not produce any changes in AST or ALT activity, which is consistent with our results (Hassan et al., 2012; Bitencourt et al., 2015). In both studies, the e�ect of CAP on carbon tetrachloride (CCl4)-induced hepa- totoxicity was investigated with discordant results. Hassan et al. (2012) found a signi�cant decrease of CCl4-induced elevation of these marker levels in rats, while Bitencourt et al. (2015) observed further elevation of liver enzyme activ- ities when CAP was administered in mice. In more recent studies, the hepa- toprotective e�ect of CAP on alcohol or ‘high fat diet’ induced liver injury in mice was described (Koneru et al., 2018; Şekeroğlu et al., 2018). Since we have not shown any e�ect of DEN on liver enzyme activity, we cannot assess the protective or harmful e�ects of CAP. Time-related DEN-induced histological changes were described in detail by Paula-Santos et al. (2014). �e duration of our experiment allowed the devel- opment of only early toxic lesions, and a longer time is needed for proliferative, dysplastic, or neoplastic lesions to development. Mohammed et al. (2014) used Fig. 4. Granular dystrophy of the renal tubules in DEN treated group. Original magni�cation: A 200×, B 400×; H&E stain (Photo. Kuchařová et al.) O rgan toxicity of diethylnitrosam ine and capsaicin in m ice – in vivo study V en du la K uc ha řo vá , O nd ře j D an ěk , M iš a Š ko rič , I va na V es el á, J ar os la va T om en en dá lo vá 44 CAP as a preventive and therapeutic agent against DEN-induced liver injury and hepatocellular carcinoma in rats with a prominent bene�cial e�ect of CAP on liver histology, which is consistent with our results. �e renal toxicity of DEN was described by Rezaie et al. (2013) with signs of acute tubular necrosis in histological sections. Our results con�rm DEN-neph- rotoxicity with the promising bene�cial e�ect of CAP. Conclusions In this study, a mild protective e�ect of CAP on DEN-induced hepatotoxicity and ne- phrotoxicity was shown only in histopathological changes. �e toxicity of CAP itself is questionable, and further studies should be performed to verify its chemopreventive potential. Acknowledgement �is study was supported by grant IGA VFU Brno 114/2016/FVL. References Basith, S., Cui, M., Hong, S., Choi, S. (2016). Harnessing the therapeutic potential of capsaicin and its analogues in pain and other diseases. Molecules, 21(8), E966. DOI: 10.3390/molecules21080966 Bitencourt, S., Stradiot, L., Verhulst, S., �oen, L., Mannaerts, I., van Grunsven, L.A. (2015). Inhibito- ry e�ect of dietary capsaicin on liver �brosis in mice. Molecular Nutrition & Food Research, 59(6), 1107–1116. DOI: 10.1002/mnfr.201400649 Bley, K., Boorman, G., Mohammad, B., McKenzie, D., Babbar, S. (2012). A comprehensive review of the carcinogenic and anticarcinogenic potential of capsaicin. 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DOI: 10.1155/2012/584728 Surh, Y.J., Lee, S.S. (1996). Capsaicin in hot chili pepper: carcinogen, co-carcinogen or anticarcinogen? Food and Chemical Toxicology, 34(3), 313–316. DOI: 10.1016/0278-6915(95)00108-5 Abstract Diethylnitrosamine (DEN) is proven to be toxic to kidneys and liver and to act as a potent carcinogen mainly in liver. Capsaicin (CAP) is an alkaloid produced by Capsicum genus plants and is considered to be a protective agent against the toxicity and carcinogenicity of many substances including DEN. �e aim of this study was to assess the toxicity of DEN and CAP in the livers and kidneys in mice. �e experiment started a�er two weeks of acclimatisation and was conducted according to the Czech animal welfare protection legal guidelines. At the end of the experiment, the mice were euthanized and the toxicity of DEN and CAP in the livers and kidneys were analysed. �e histopathological examination of the livers revealed multifocal lymphoplasma- cytic reaction in parenchyma in the DEN treated group. CAP used as both a preventive and therapeutic agent caused a reduction in the number and extent of lesions. In the CAP group, mitotic �gures were found, indicat- ing xenobiotic-induced hepatotoxicity or regenerative changes. In the kidneys, DEN also revealed multifocal lymphoplasmacytic reactions that have been mitigated by CAP. Moreover, histopathological observation of the kidneys in the DEN group revealed granular dystrophy of the renal tubules, which were not presented in CAP treated mice. Levels of ALT, AST activity, total protein, and albumin concentrations were not statis- tically di�erent among control and experimental groups. In this study, the mild protective e�ect of CAP on DEN-induced hepatotoxicity and nephrotoxicity was shown only in histopathological changes. �e toxicity of CAP itself is questionable, and further studies should be performed to verify its chemopreventive potential. Key words: capsaicin, diethylnitrosamine, hepatotoxicity, mice, nephrotoxicity Received: [2018.06.07] Accepted: [2018.11.21] O rgan toxicity of diethylnitrosam ine and capsaicin in m ice – in vivo study V en du la K uc ha řo vá , O nd ře j D an ěk , M iš a Š ko rič , I va na V es el á, J ar os la va T om en en dá lo vá 46 Toksyczność narządowa dietylo-nitrozaminy i kapsaicyny u myszy – badanie in vivo Streszczenie Udowodniono, że dietylo-nitrozamina (DEN) działa toksycznie na nerki i wątrobę oraz jako silny czynnik rakotwórczy, głównie w wątrobie. Kapsaicyna (CAP) jest alkaloidem wytwarzanym przez gatunki z rodzaju Capsicum i jest uważana za czynnik ochronny przeciwko toksyczności oraz rakotwórczości wielu substancji, w tym również DEN. Celem tego badania była ocena toksyczności DEN i CAP w wątrobie oraz nerkach my- szy. Eksperyment rozpoczął się po dwóch tygodniach aklimatyzacji i został przeprowadzony zgodnie z cze- skimi przepisami prawnymi dotyczącymi ochrony zwierząt. Pod koniec doświadczenia myszy uśmiercano, następnie dokonano oceny toksyczności DEN i CAP w wątrobie oraz nerkach. Badanie histopatologiczne wątroby wykazało wieloogniskową reakcję limfoplazmatyczną w miąższu w grupie poddanej działaniu DEN. CAP stosowany jako środek zapobiegawczy i leczniczy powodował zmniejszenie liczby oraz roz- miaru zmian. W grupie poddanej CAP stwierdzono formy mitotyczne wskazujące na hepatotoksycz- ność wywołaną ksenobiotykami lub też na zmiany regeneracyjne. W nerkach poddanych DEN stwier- dzono również wieloogniskową reakcję limfoplazmatyczną, która była łagodzona przez CAP. Ponadto obserwacja histopatologiczna nerki w grupie DEN ujawniła dystro�ę ziarnistą kanalików nerkowych, której nie przedstawiono u  myszy poddanych CAP. Poziomy aktywności ALT, AST, stężenia białka i albuminy nie były statystycznie różne w grupach kontrolnych i eksperymentalnych. W badaniu tym łagodny ochronny wpływ CAP na indukowaną przez DEN hepatotoksyczność i nefrotoksyczność wykaza- no tylko w zmianach histopatologicznych. Toksyczność samej CAP jest wątpliwa i należy przeprowadzić dalsze badania w celu zwery�kowania jej potencjału chemoprewencyjnego. Słowa kluczowe: kapsaicyna, dietylo-nitrozoamina, hepatotoksyczność, myszy, nefrotoksyczność Information on the authors Vendula Kuchařová She is a PhD student and assistant at the Department of Physiology at the University of Veterinary and Pharmaceutical Sciences Brno. �e topic of her PhD study is �e e�ect of capsaicin on tumour progression – in vivo model. Ondřej Daněk He studies in the Master’s Degree Study Programme at the Faculty of Veterinary Medicine at the Universi- ty of Veterinary and Pharmaceutical Sciences Brno. He is interested in cell biology and animal physiology. Miša Škorič He is an associate professors and leader of the discipline “Pathological Morphology” at the Department of Pathological Morphology and Parasitology at the University of Veterinary and Pharmaceutical Sciences Brno. He is a member of ESVP (European Society of Veterinary Pathologists), and his research activities include the Mycobacterioses of animals, the detection of tissue defects in transplanted pulmonary valve (porcine model), the repair of bone defects, and colorectal anastomoses (rabbit). Ivana Veselá She is an assistant professor at the Department of Physiology at the University of Veterinary and Pharma- ceutical Sciences Brno. She is interested mainly in cell biology and natural substances such as aerugino- sin-865, resveratrol, and capsaicin in medicine. Jaroslava Tomenendálová She is an assistant professor and leader of the discipline “Pathological Physiology” at the Department of Physiology at the University of Veterinary and Pharmaceutical Sciences Brno. She also focuses on cell biology and natural products, predominantly in cancer prevention and therapy.