The effects of Cyclosporin A and Heteropterys tomentosaon the rat liver

An Acad Bras Cienc Anais da Academia Brasileira de Ciências An. Acad. Bras. Ciênc. 0001-3765 1678-2690 Academia Brasileira de Ciências A Ciclosporina A (CsA) é uma droga imunossupressora amplamente utilizada que está associada a diversos efeitos colaterais, dentre eles hepatotoxicidade. Heteropterys tomentosa é uma planta brasileira eficiente na redução dos danos causados pela CsA nos testículos e próstata de ratos. O objetivo deste estudo foi avaliar o efeito da CsA e H. tomentosa(administrados isolada ou simultaneamente) no fígado de ratos Wistar. Os animais foram tratados diariamente com água (controle), CsA (15mg/kg/dia), infusão de H. tomentosa ou CsA+H. tomentosa, por 21 ou 56 dias. Os tratamentos não alteraram a morfologia do fígado nem causaram fibrose. A administração de H. tomentosa por 21 dias aumentou o número de núcleos de hepatócitos e proporção volumétrica de células de Kupffer. Após 56 dias de tratamento, a administração de H. tomentosa não alterou os parâmetros analisados. As dosagens bioquímicas plasmáticas e a estereologia do fígado revelaram danos causados pelo tratamento com CsA por 21 dias; estes resultados não foram observados após 56 dias de tratamento. O tratamento simultâneo com CsA e H. tomentosa por 21 e 56 dias não atenuou ou acentuou os efeitos hepáticos da CsA. O presente estudo revelou que o tratamento com a CsA por 21 dias causou mais alterações ao fígado que o tratamento durante 56 dias; isso pode estar relacionado a recuperação hepática após o tratamento a longo prazo. INTRODUCTION Cyclosporin A (CsA) is a neutral, hydrophobic, cyclic peptide with 11 amino acids (Shevach 1985) that is administered as an immunosuppressant drug (Shevach 1985, Matsuda and Koyasu 2000). This drug is widely used to treat autoimmune diseases, being associated with reduction in the number of hospitalization days for transplanted patients and for rejection episodes (Rezzani 2004). However, several side effects are associated with CsA treatment such as: nephrotoxicity (Bertani et al. 1987, Baliga et al. 1997, Fellstrom 2004, Rezzani 2004, Hagar et al. 2006, Josephine et al. 2006, 2007, Rezzani et al. 2006b), hepatotoxicity (Durak et al. 2004, Hagar 2004, Rezzani 2004, 2006, Kurus et al. 2008, Bohmer et al. 2011), testicular dysfunction (Monteiro et al. 2008) and damages to the ventral prostate (Freitas et al. 2012a). Most of these are related to the increase of reactive oxygen species (Wolf et al. 1997, Buffoli et al. 2005, Rezzani 2006), causing damage to tissue structure and functional disorders (Whiting et al. 1983). Several researchers have investigated subs tances that reduce CsA side effects (Rezzani et al. 2001, 2006a, b, Parra Cid et al. 2003, Monteiro et al. 2008, Wongmekiat et al. 2008). Heteropterys tomentosa A. Juss. (sin. Heteropterys aphrodisiaca O. Mach.), a native species from the Brazilian "Cerrado" (a savannah-like biome), is used by the local population as a hypoglycemiant to treat diabetes (Guarim Neto and Morais 2003, Macedo and Ferreira 2004), as an aphrodisiac and to treat nervous weakness and collapse (Corrêa 1984, Pott and Pott 1994). The antioxidant properties of H. tomentosa root extract (BST0298) were demonstrated by Mattei et al. (2001), and this same extract was also efficient in the recovery of learning and memory of aged rats (Galvão et al. 2002). H. tomentosa root infusion has been successfully used to reduce CsA-related effects on testicular tissue (Monteiro et al. 2008), ventral prostate and biochemical parameters related to hepatotoxicity - plasma levels of glutamic oxaloacetic transaminase, total and indirect bilirubin and cholesterol (Freitas et al. 2013). Considering the well documented hepatic damage caused by CsA, which is probably due to oxidative stress, and the potential of H. tomentosa as an antioxidant, we investigated the potential benefits of using this medicinal plant against the CsA-related hepatotoxicity. The following aspects were studied: (1) determination of the side effects of CsA on the liver structure and function; (2) evaluation of hepatotoxicity caused by H. tomentosa intake; (3) effects of simultaneous treatment with CsA and H. tomentosa. MATERIALS AND METHODS PREPARATION OF THE TREATMENT SOLUTIONS The methodology used for infusion preparation was the same as that used by previous authors (Monteiro et al. 2008, 2011, Gomes et al. 2011, Freitas et al. 2012b, 2013). In brief, Heteropterys tomentosa A. Juss. (sin. Heteropterys aphrodisiaca O. Mach., herbarium CPAP number 21625) roots were obtained from plants cultivated by Embrapa Pantanal in Corumbá, MS, Brazil. The plants were grown in controlled conditions of soil, fertilization and irrigation, without insecticides. The roots were harvested, dried and ground. The infusion was prepared immersing ground roots (75.76mg/kg of body weight) in 100mL of boiled water; the animals' weight was used to calculate quantity of ground roots. The infusion was maintained at room temperature for 4 hours, and then filtered. It was kept at 4° C and used for two days. Cyclosporin A (Sandimmun Neoral(r), Novartis Pharma AG, Switzerland) was administered in a therapeutic dose (15mg/kg body weight) as in previous studies (Chung et al. 2005, Kotolová et al. 2006, Monteiro et al. 2008, Bohmer et al. 2011, Freitas et al. 2012a, 2013). EXPERIMENTAL ANIMALS AND PROTOCOL This experimental protocol was carried out according to the standards of the Brazilian Society for Laboratory Animals Sciences (SBCAL/COBEA) and was approved by the Ethics Committee for Animal Studies of the State University of Campinas (Protocol number 2295-1). The treatment protocol was similar to that previously employed (Monteiro et al. 2008, Freitas et al. 2013). Forty Wistar rats (Rattus norvegicus albinus), eight weeks old, were obtained from the Multi-research Center for Biological Investigation of Laboratory Animal - CEMIB (State University of Campinas, Campinas - SP, Brazil) and maintained under standard conditions with 12h light/dark periods, fed with commercial chow and water "ad libitum". They were separated into two groups of 20 animals each. One group was treated for 21 days and the other for 56 days. In each group, the animals were separated in 4 subgroups (n=5 in each): Control groups (CG) received 0.5mL of distilled water; CsA-treated groups (CsA) received CsA diluted in 0.5 mL distilled water; H. tomentosa treated groups (Ht) received 0.5mL of infusion; and groups treated with CsA and H. tomentosa (CsA+Ht) received the CsA dose diluted in 0.5 mL of infusion. All treatments were administered daily, by gavage. The administrations of CsA for 21 (Buffoli et al. 2005, Rezzani et al. 2005a, b, Türk et al. 2007) or 56 days (Monteiro et al. 2008, Freitas et al. 2012a, 2013) were chosen based on previous experiments reported in literature. COLLECTION OF THE BIOLOGICAL SAMPLES The animals were weighed and anesthetized with Xylazine and Ketamine (5 and 80 mg/kg, respectively) by intramuscular injection. Blood was collected from the left ventricle by cardiac puncture using Vacuette(r) tubes with heparin, centrifuged at 3500rpm for 10 minutes (4° C) and the plasma was collected for further biochemical analyses. The liver was collected and weighed. Liver relative weight (%) was calculated using the formula: (Liver weight x 100)/ body weight. PLASMA BIOCHEMISTRY The plasma was used to quantify the levels of glucose (Kit Laborlab(r), enzymatic and colorimetric methods), triglycerides (Kit Bioclin(r), enzymatic and colorimetric methods), total cholesterol (Kit Laborlab(r), enzymatic and colorimetric methods), total proteins (by refractometry, analysis made at VetPat Laboratory), creatinine (Kit Bioclin(r) Kit, colorimetric and kinetic methods), alkaline phosphatase (Kit Laborlab(r), kinetic method), glutamic pyruvate transaminase/aspartate amino transferase (GPT/AST) (Kit Bioclin(r), colorimetric method) and glutamic oxaloacetic transaminase/alanine aminotransferase (GOT/ALT) (Kit Bioclin(r), colorimetric method). LIGHT MICROSCOPY Liver fragments were fixed in 4% paraformaldehyde (in phosphate buffer 0.1M, pH 7.4) for 24 hours, at room temperature. The fragments were washed in the same buffer, dehydrated in ethanol, cleared in xylol and embedded in paraffin (Histosec(r), Leica). The tissue was sectioned with 5µm thickness. One slide was stained with hematoxylin and eosin for morphological, morphometrical and stereological analyses. Another slide was stained with Masson's trichrome (EasyPath(r) kit) for fibrosis detection. MORPHOMETRICAL AND STEREOLOGICAL ANALYSES The proportions of hepatocytes (nuclei and cytoplasm), sinusoid capillaries and Kupffer cells were estimated using a grid with 130 intersections over digital images (40x objective). Ten images were analyzed per animal. The number and diameter of all hepatocyte nuclei were counted and measured, respectively, in 10 fields per animal (40x objective), in which each field had 2.97x104 µm2. The number of hepatocyte nuclei (HN) per µm2 was calculated (HN/ µm2 = Number of Hepatocytes per field / field area). Hepatocyte nuclei volume (NV) was calculated using the formulas: area=πr2 and volume= 4/3πr3. The hepatocyte cytoplasm volume (CV) was calculated using the formula: CV = (NV x cytoplasm proportion)/nuclei proportion. The hepatocyte volume was the sum of nuclei and cytoplasm volumes. STATISTICAL ANALYSES A nonparametric test (Kruskal Wallis Multiple Comparison) was performed using the software Minitab 16. For all the tests the significance was 95%. All values are shown as mean ± standard deviation (SD). RESULTS CYCLOSPORIN A - EFFECTS ON LIVER CsA treatment did not alter the liver absolute and relative weight (Figure 1). The tissue structure did not show any alteration either (Figures 2 and 3) and fibrosis was not observed even after 56 days of treatment. Figure 1 - Liver absolute (g) and relative weight (%). Control, treated with CsA (CsA), Heteropterys tomentosa (Ht) and CsA + H. tomentosa (CsA+Ht), for 21 or 56 days. Figure 2 - Liver (H&E staining). Control (A), treated with CsA (B), H. tomentosa infusion (C) and both substances (D), for 21 days. Hep: hepatocytes; white arrows: Kupffer cells; black arrows: sinusoids. Bars= 50µm. Figure 3 - Liver (H&E staining). Control (A), treated with CsA (B), H. tomentosa infusion (C) and both (D) for 56 days. Hep: hepatocytes; white arrows: Kupffer cells; black arrows: sinusoids. Bars= 50µm. Volumetric proportions of hepatocytes were reduced in the group treated with CsA for 21 days (p=0.045); and the volumetric proportions of sinusoids increased 29.06%, however this alteration was not statistically significant. These results were not observed after treatment with CsA for 56 days (Table I). Table I Hepatic parameters of Wistar rats treated with water (control), CsA, H. tomentosa infusion or CsA+H. tomentosa infusion for 21 or 56 days. Parameters Groups 21 Days 56 Days CG CsA Ht CsA+Ht CG CsA Ht CsA+Ht Volumetric Proportions (%) Hepatocytes (% nuclei; % cytoplasm) 89.15 ± 2.39a (11.39 ± 1.28a; 88.61 ± 1.28a) 86.17 ± 2.61b (12.22 ± 0.69a; 87.78 ± 0.69a) 88.48 ± 1.08ab (15.19 ± 1.69b; 84.81 ± 1.69b) 90.46 ± 1.50a (12.24 ± 2.37ab; 87.76 ± 2.37ab) 90.09 ± 2.04 (11.85 ± 1.67; 88.15 ± 1.67) 90.26 ± 2.83 (12.72 ± 1.89; 87.28 ± 1.89) 89.01 ± 4.64 (11.88 ± 1.50; 88.12 ± 1.50) 88.71 ± 0.85 (12.12 ± 2.04; 87.88 ± 2.04) Sinusoids 9.43 ± 2.38ab 12.17 ± 2.87a 9.14 ± 1.06ab 7.01 ± 1.26b 8.06 ± 1.56 7.37 ± 3.14 8.71 ± 4.49 8.85 ± 1.09 Kupffer cells 1.41 ± 0.26a 1.65 ± 0.44a 2.38 ± 0.37b 2.52 ± 0.33b 1.85 ± 0.51 2.37 ± 0.45 2.28 ± 0.39 2.45 ± 0.33 Volume (μm3) Hepatocyte 2501.46 ± 145.0a 2267.7 ± 313.1ab 1760.0 ± 242.5c 1887.9 ± 281.0bc 2265.9 ± 429.7 2111.2 ± 205.3 2322.5 ± 468.4 2525.1 ± 419.7 HC 2217.9 ± 161.0a 1991.9 ± 286.5ab 1491.8 ± 198.1c 1661.73 ± 286.6bc 2002.7 ± 416.3 1844.96 ± 210.8 2050.3 ± 439.4 2225.0 ± 418.7 HN 283.55 ± 16.67a 275.80 ± 29.40a 268.12 ± 56.89ab 226.19 ± 21.23b 263.18 ± 20.25a 266.30 ± 26.53a 272.22 ± 38.02ab 300.05 ± 25.85b Number of HN per area - µm2 (x10-3) 1.00 ± 0.09a 1.01 ± 0.13a 1.23 ± 0.13b 1.07 ± 0.16ab 0.94 ± 0.10a 1.03 ± 0.2ab 1.00 ± 0.14ab 1.11 ± 0.01b Control group (CG), Cyclosporin A treated group (CsA), Heteropterys tomentosa treated group (Ht) and group with simultaneous treatment of CsA and H. tomentosa (CsA+Ht). HC: Hepatocyte cytoplasm; HN: Hepatocyte nucleus. The values are mean ± SD. In each row, values with different superscripts are significantly different (p The treatment with CsA for 21 days increased total cholesterol (p=0.021) and in comparison to the control group (Table II). GOT levels were 40.38% higher in the CsA group when compared to the control, however the difference was not statistically significant (p=0.06). Yet, the treatment with CsA for 56 days increased GPT levels (p=0.012) (Table II). Table II Plasma biochemical parameters of Wistar rats treated with water (control), CsA, H. tomentosa infusion or CsA+H. tomentosa infusion for 21 or 56 days. Parameters Groups 21 Days 56 Days CG CsA Ht CsA+Ht CG CsA Ht CsA+Ht Glucose (mg/dL) 272.13 ± 43.46a 276.96 ± 38.77a 301.26 ± 44.94ab 367.78 ± 67.54b 305.22 ± 45.02 317.39 ± 75.93 300.39 ± 75.67 293.09 ± 35.36 Total Cholesterol (mg/dL) 93.29 ± 5.45a 105.42 ± 7.82b 91.87 ± 6.35a 103.87 ± 4.08b 104.26 ± 5.21a 108.13 ± 14.58ab 101.03 ± 7.12a 114.45 ± 0.07b Triglycerides (mg /dL) 109.31 ± 7.91ab 105.37 ± 9.29ab 89.13 ± 25.38a 124.86 ± 22.70b 124.77 ± 19.07 142.02 ± 22.17 123.07 ± 27.89 148.53 ± 30.34 Alkaline Phosphatase (U/L) 109.27 ± 32.12ab 129.43 ± 15.40a 72.19 ± 49.68b 124.67 ± 101.20ab 86.72 ± 19.17 81.03 ± 23.94 76.45 ± 19.63 97.9 ± 43.92 Total Proteins (g/dL) 6.3 ± 0.2 6.55 ± 0.44 6.45 ± 0.38 6.35 ± 0.25 7.44 ± 0.46 6.92 ± 0.48 7.4 ± 0.58 7.15 ± 0.17 GOT (U/L) 81.21 ± 20.76ab 114.21 ± 27.43a 71.36 ± 8.59b 81.19 ± 18.60b 86.57 ± 14.74 91.79 ± 38.12 81.64 ± 12.67 82.43 ± 21.94 GPT (U/L) 41.73 ± 6.26 43.22 ± 11.32 47.14 ± 12.75 46.09 ± 7.74 39.09 ± 3.50a 57.82 ± 14.29bc 44.23 ± 5.84ab 61.04 ± 5.87c Creatinine (mg/dL) 0.65 ± 0.11a 0.66 ± 0.06a 0.78 ± 0.09ab 0.79 ± 0.09b 0.75 ± 0.07 0.80 ± 0.20 0.73 ± 0.10 0.75 ± 0.10 Control group (CG), Cyclosporin A treated group (CsA), Heteropterys tomentosa infusion treated group (Ht) and group with simultaneous treatment of CsA and H. tomentosa (CsA+Ht). GOT: Glutamic Oxalacetic Transaminase; GPT: Glutamic Pyruvate Transaminase. The values are mean ± SD. In each row, values with different superscripts are significantly different (p Heteropterys tomentosa - EFFECTS ON THE LIVER The treatment with H. tomentosa (during 21 and 56 days) did not alter liver absolute and relative weights (Figure 1) or the morphology (Figures 2 and 3) and nor did it cause fibrosis. However, an increase of the volumetric proportion of Kupffer cells was observed after treatment with the infusion for 21 days (p=0.013). Furthermore, these animals showed an increase of hepatocyte nucleus proportion and a reduction of their cytoplasm (p=0.003) (Table I). Higher number of hepatocyte nuclei was found per µm2 (p=0.023). The treatment with H. tomentosa also reduced the total and the cytoplasmic volume of hepatocytes (p=0.002 and 0.001 respectively) (Table I). No alterations of hepatic morphometry/stereology and plasma parameters were observed after the treatment with H. tomentosa for 56 days (Tables I and II). CSA AND H. tomentosa ASSOCIATED - EFFECTS ON THE LIVER As observed in the other groups, simultaneous treat ment with CsA and infusion did not alter liver absolute and relative weights (Figure 1) or morphology (Figures 2 and 3) and did not cause fibrosis. The group CsA+Ht (21 days) did not show the alterations observed for the CsA group (21 days) (Table I). On the other hand, in this group the volumetric proportion of Kupffer cells increased (p=0.002). This was also observed for animals treated only with H. tomentosa (during 21 days). Higher hepatocyte nuclear volume was observed when compared to the control and the CsA group (21 days) (p=0.004) (Table I). As observed for the Ht group, the simultaneous treatment with CsA and H. tomentosa also caused reduction of hepatocyte volume (p=0.012), due to the reduction on cytoplasm volume (p=0.012) (Table I). In the CsA+Ht group (21 days) the levels of GOT did not differ significantly in relation to the control or the CsA group (Table II). The plasma creatinine levels were higher in CsA+Ht group (21 days) when compared to the control (p=0.049) and CsA (p=0.02) (21 days) (Table II). Despite the results observed after 21 days of treatment, animals treated with both infusion and CsA for 56 days showed an increase in the number of hepatocyte nuclei per µm2 (p=0.04) (Table I). Total cholesterol and GPT levels were increased in CsA+Ht group (56 days) when compared to the control (p=0.044 and 0.001, respectively) (Table II). DISCUSSION Hepatotoxicity induced by CsA administration is widely reported in literature (Wolf et al. 1997, Durak et al. 2004, Hagar 2004, Rezzani 2004, 2005a, 2006, Kurus et al. 2008, Bohmer et al. 2011). There are two possible mechanisms of CsA-induced hepatotoxicity: (1) due to an excess of free radical production during CsA metabolism in the liver; or (2) CsA may impair the antioxidant defense (Durak et al. 2004). Rezzani et al. (2005a) suggested that the oxidative stress induced by CsA is associated with the imbalance of free oxygen radical production and the antioxidant defense system. The increase of CsA-associated oxidative stress is positively correlated with biochemical parameter alterations, which are characteristic of hepatotoxicity (Hagar 2004). According to Bohmer et al. (2011), CsA-associated histopathological changes on the liver comprise sinusoidal dilatation, hepatocyte cyto plasmic vacuolization, cell infiltration, parenchymal mitosis and moderate hepatocellular necrosis. Rezzani et al. (2005a) observed evident liver morphological alterations caused by CsA (15mg/kg/day) subcutaneously injected in Wistar rats, for 30 days. These authors observed widespread cell swelling and congestion of sinusoids. Hagar (2004) observed, in the rabbits' liver, severe hydropic degeneration in parenchymal cells after treatment with CsA (25mg/kg/day, orally during 10 days). However, Bohmer et al. (2011), working with CsA treated animals (5 and 15mg/kg daily) by gavage for 8 weeks, did not observe clear histopathological and clinicopathological alterations of the liver, which supports our findings. The absence of hepatic histopathological impairment observed in this study can be related to the dose and route of drug administration. Despite the absence of clear morphological alteration, the treatment with CsA for 21 days reduced the volumetric proportion of hepatocytes and increased (not statistically significant) the volumetric proportion of sinusoids. Bohmer et al. (2011) listed sinusoids dilatation as one of the histopathological changes associated with CsA administration. However, the authors did not explain how CsA induced this change. CsA-associated fibrosis is largely reported in the kidney (Bakker et al. 2003, Ling et al. 2003, Li et al. 2004). Hagar (2004) working with CsA (25mg/kg daily), administered daily to rabbits, observed mild connective tissue proliferation in the liver periportal region. However, in the present study, CsA did not cause fibrosis even after 56 days of treatment, which was probably due to the lower CsA-dose used in our experiment. The histopathological alterations were consistent with the biochemical findings. CsA administered for 21 days caused increase of total cholesterol and GOT levels. On the other hand, the treatment with CsA for 56 days caused an increase of GPT levels. Bohmer et al. (2011) did not observe alterations of GOT and GPT levels after treatment of rats with 15mg/kg/day of CsA by gavage for 30 days. According to the authors, CsA caused immunosuppression without hepatotoxicity. The treatment with CsA is known to cause hyperglycemia (Neto et al. 1999, Freitas et al. 2012a) however, in our study this response was not observed even after treatment for 56 days. The simultaneous treatment with H. tomentosa and CsA caused an increase of glucose and creatinine level after 21 days of treatment; these effects were not observed after a long term treatment or when the animals were treated with only CsA or H. tomentosa. The increased volumetric proportion of hepa tocyte nuclei associated with the infusion treatment can be explained by the higher number of hepatocyte nuclei (hyperplasia) since an increase of nucleus area or volume (hypertrophy) was not observed. The increase of hepatocyte nucleus number and Kupffer cells proportion could indicate toxicity induced by H. tomentosa after the treatment for 21 days. The effect of the infusion administration can be associated with the acute treatment since after 56 days of treatment no alterations caused by the plant could be observed. The proliferation of hepatocytes could be related to liver recovery after injury (Tan et al. 2014). The use of antioxidant substances against CsA-induced hepatotoxicity is reported in literature. The efficiency against CsA-induced hepatotoxicity of taurine (Hagar et al. 2006), oral L-arginine (Kurus et al. 2008), melatonin (Rezzani et al. 2005a) and vitamins C and E (Durak et al. 2004) has been confirmed. According to Ng et al. (2000) flavonoid compounds exhibit antioxidant and free radical scavenging properties. Flavonoids are also considered as a protective substance against harmful external influences (Hassig et al. 1999). Galvao et al. (2002) listed flavonoid glycosides among the substances present on H. tomentosa root extract. However, according to Galvao et al. (2002) it is not possible to affirm if the effects of H. tomentosa could be attributed to one or more chemical constituents. In the present study the treatment with H. tomentosa together with CsA for 21 days did not alter sinusoids and hepatocyte proportions, as found for the treatment with CsA (21 days). In addition, the alteration observed in GOT levels after the treatment with CsA (21 days) was not present for animals treated simultaneously with the drug and the infusion. However, the simultaneous treatment (for 21 days) caused alterations of glucose and creatinine levels. The alteration of GPT levels caused by CsA (56 days) also occurred in the CsA+Ht group. The absence of hepatotoxicity after 56 days of CsA administration did not allow the evaluation of H. tomentosaprotection. The simultaneous treatment administered for 56 days did not induce hepatotoxicity. Freitas et al. (2013), working with Wistar rats treated with CsA, H. tomentosa and both substances, observed an increase of GOT, bilirubin (total and indirect), triglycerides, cholesterol and glucose levels caused by CsA. They also observed that animals treated simultaneously with CsA and infusion showed normalization of GOT, triglycerides and glucose levels. The above authors did not observe any alterations caused by H. tomentosa on biochemical parameters of hepatotoxicity. Freitas et al. (2013) worked with 90 days-old Wistar rats and the animals were treated for 56 days with the same dose of CsA and H. tomentosa used in the present study. The differences between the present results and the findings of Freitas et al. (2013) could be due to the time of drug administration (in the case of the animals treated for 21 days), the different source of H. tomentosa roots, animals' initial age (we started the experiment with 56 days-old animals) or the animals' individual sensibility to the treatment. CONCLUSIONS The results suggested that the treatment with CsA for 21 days caused hepatotoxicity which was reduced over a longer administration period (56 days). Possibly the hepatic defense system against higher oxidative stress was able to recover during this period with an increase of the enzymes involved in free radical elimination. H. tomentosa caused alterations of some of the parameters analyzed, mainly after 21 days of treatment. The association with the plant administration and hepatotoxicity has to be better studied. 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