The effects of Cyclosporin A and Heteropterys tomentosa on the rat liver

Cyclosporin A (CsA) is a widely employed immunosuppressive drug that is associated with several side effects, among then hepatotoxicity. Heteropterys tomentosa is a Brazilian plant efficient in reducing damage caused by CsA on the rat testis and prostate. The aim of this study was to evaluate the effect of CsA and H. tomentosa (administered isolated or simultaneously) on the liver of Wistar rats. The animals were treated daily with water (control), CsA (15mg/kg/day), H. tomentosa infusion or CsA+H. tomentosa, for 21 or 56 days. The treatments did not alter liver morphology or cause fibrosis. H. tomentosa administered for 21 days increased the number of hepatocyte nuclei and Kupffer cell volumetric proportion. After 56 days of treatment, H. tomentosa administration did not alter the parameters analyzed. Biochemical plasma dosages and liver stereology showed impairment caused by CsA-treatment after 21 days; these results were not observed after 56 days of treatment. The simultaneous treatment with CsA and H. tomentosa for 21 or 56 days did not alleviate nor accentuate CsA hepatic effects. The present study showed that the 21 days treatment with CsA caused more alteration to the liver than the 56 days treatment; this could be related to hepatic recovery after the long term treatment.

Several researchers have investigated substances 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 andMorais 2003, Macedo andFerreira 2004), as an aphrodisiac and to treat nervous weakness and collapse (Corrêa 1984, Pott andPott 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 hepatotoxicityplasma 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.

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.

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 Multiresearch Center for Biological Investigation of Laboratory Animal -CEMIB (State University of Campinas, Campinas -SP, Brazil) and maintained under standard conditions with 12h light/dark CYCLOSPORIN A AND H. tomentosa ON THE LIVER 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.

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 ® 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.

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 ® , 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 ® 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 2.97x10 4 µm 2 .The number of hepatocyte nuclei (HN) per µm 2 was calculated (HN/ µm 2 = Number of Hepatocytes per field / field area).Hepatocyte nuclei volume (NV) was calculated using the formulas: area=πr 2 and volume= 4/3πr 3 .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).

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.
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).
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).

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 µm 2 (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 treatment 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 µm 2 (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 CsAinduced 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), CsAassociated histopathological changes on the liver comprise sinusoidal dilatation, hepatocyte cytoplasmic 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 hepatocyte 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 CsAinduced 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. tomentosa protection.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 CYCLOSPORIN A AND H. tomentosa ON THE LIVER 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.The simultaneous treatment with CsA and H. tomentosa did not accentuate nor attenuate the CsA-related side-effects studied.

ACKNOWLEDGMENTS
The authors wish to thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant number 2011/01160-6) for the financial support.They are also grateful to Enzo Guidotti Mattioli for the Statistical support.The authors declare that there are no conflicts of interest.This article is part of a PhD thesis.

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.
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<0.05) by Kruskal Wallis Multiple Comparison test.

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.
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<0.05) by Kruskal Wallis Multiple Comparison test.