Protective Effect of Vitamin E against Moxifloxacin Induced Side Effect in Rats

| Our investigation was carried out to assess the protective effect of vitamin E against the moxifloxacininduced side effects in rats. A total of 80 adult male albino rats were allocated into four equal groups. The first group represented as a control and given physiological saline. The second and third groups were orally received vitamin E (18 mg/kg BW) and moxifloxacin (7.2 mg/kg BW), respectively for 21 successive days. The fourth group was received Vit E concurrently with moxifloxacin for 21 successive days. Administration of moxifloxacin to albino rats resulting in a significant increase in the activities of ALT (Alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase) beside significant decrease in the levels of total serum protein and albumin, in addition to an elevation in serum creatinine and urea all over the experimental period compared with control group. In addition, a significant decrease in catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) as well as a significant elevation of malondialdehyde (MDA) concentration were recorded in moxifloxacin-treated group when compared with the control group. The histopathological alterations in liver and kidney of the examined rats confirmed the obtained biochemical results. Co-administration of moxifloxacin with Vit E for 21 successive day resulting in a significant reduction of the activities of liver enzymes, creatinine and urea, while serum total proteins, albumin and globulins turned toward the normal levels beside an elevation of CAT, SOD and GPx with a significant decrease in the concentration of MDA when compared with moxifloxacin alone-treated group. Our results revealed that Vit E could protect against moxifloxacin-induced side effects. Therefore, Vitamin E should be taken with moxifloxacin to decrease its adverse effects.


INTRODUCTION
T he fluoroquinolone antimicrobials are utilized in the medication of a wide number of bacterial diseases however their therapeutic use has been associated with various toxicological effects (Leone et al., 2003;Owens and Ambrose, 2005;Thompson, 2007;de Guidi, 2011). The mechanism of action of fluoroquinolones involves inhibition of deoxyribonucleic acid (DNA) gyrase (topoisomerase II) and (topoisomerase IV), enzymes involved in bacterial DNA replication, transcription, repair, and recombination (Thompson, 2007;Sinbad et al., 2019).
Moxifloxacin is a fourth-generation of fluoroquinolone antimicrobial, (at first called BAY 12-8039) and it is advertised worldwide under the brand name avelox. Moxifloxacin is active against both Gram-positive and Gram-negative bacteria. The moxifloxacin is a promising new agent that may have added to existing antituberculotic agents by evaluating the action of moxifloxacin in tuberculosis treatment (Minov et al., 2018). and adults. Other unfriendly impacts include deleterious effects on sensory system, liver and kidney functions, etc., (Committee of infectious diseases, 2006). Vitamin E is a term corresponding to a small group of tocopherols of which α-tocopherol is the most bioactive, being also considered to be the most potent antioxidant of the lipid soluble form, responsible for the protection of membrane polyunsaturated fatty acid against lipid peroxidation (Carletti et al., 2007;Eraslan et al., 2007). Vitamin E primarily induces an antioxidant effect and is known to be the compound with the highest biological activity. Vitamin E acts as a free radical scavenger in the prevention of diseases and thereby inhibits lipid peroxidation (El-Demerdash, 2004).
The present study was designed to assess the (i) adverse effects of moxifloxacin and (ii) protective effect of vitamin E as antioxidants through investigation of some liver and kidney function tests beside oxidant/antioxidant status and histopathological changes in liver and kidney.

DruGS
Moxifloxacin tablet (avelox ® , 400 mg), was obtained from Future pharmaceutical industries. Human therapeutic dose of moxifloxacin converted to rat dose according to (Paget and Barnes, 1964). Vitamin E (capsule, 1000 mg) was purchased from PHARCO pharmaceutical industries CO., Alex., Egypt.) and was dissolved in corn oil.

animalS
Eighty adult male albino rats weighed 150 to 170g were used in the study. They were purchased from laboratory animal farm, Faculty of Veterinary Medicine, Zagazig University. They were housed in polypropylene cages in a temperature and humidity-controlled room. All animals were allowed access to food, water ad libitum. Animal housing and care and the experimental protocols were conducted as stipulated in the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH) and as approved by the local authorities of Zagazig University, Zagazig, Egypt. All efforts were made to minimize animal suffering. They were kept for two weeks before being used in the study.

ExpErimEntal DESiGn
Before dose administration, the body weight of each animal was determined and the dose was calculated according to the body weight. Rats were classified into 4 groups each contain 20 rats, 1 st group (control), rats in this group were not medicated and received normal saline. Second group (Vit. E), rats in this group received repeated oral doses of vitamin E (100 mg/kg BW once daily) for 21 successive days (El Maghraby and Taha, 2012) as a standard antioxidant. Third group (Moxifloxacin), rats received a repeated oral dose of moxifloxacin (7.2 mg/kg BW once daily) for successive 21 days (Pardillo et al., 2008). Fourth group (Moxifloxacin -Vitamin E) rats in this group received repeated oral doses of Moxifloxacin (7.2 mg/kg BW) concurrently Vitamin E (100 mg/kg BW) once daily for 21 days.

prEparation of SErum SamplE anD tiSSuE SamplE
Rats were sacrificed and blood samples were collected in a sterile Wassermann tube without anticoagulant from 5 rats/ group on the 1 st , 7 th , 14 th and 21 st days post treatment and allowed to clot for 30 minutes and serum was separated by centrifugation at 3000 rpm for 15 minutes, the top yellow layers of serum were pipette off without distributing the white buffy layer. Serum was stored at -20ºC in Eppendorf tubes till the determination of serum biochemical analysis. Liver and kidney of each rat were collected on 7 th and 14 th days post treatment. They were kept in 10% neutralbuffered formalin for histopathological examination.

DEtErmination of livEr anD kiDnEy function
The preserved serum used for determination of the activities of alanine aminotransferase (ALT), and aspartate aminotransferase (AST) (Tietz, 1976), alkaline phosphatase (ALP) (Belfield and Goldberg, 1971), creatinine (Henry et al., 1974), urea (Vassault et al., 1986), total protein and albumin (Gassbaro et al., 1972). Serum globulin was calculated by subtraction of the obtained albumin level from total protein level.

hEpatic anD rEnal hiStopatholoGical Evaluation
Liver and kidney tissues were kept in 10% neutral-buffered formalin for 24 h at that point tissue handling and paraffin blocks preparation were done (Suvarna et al., 2013).

StatiStical analySiS
The obtained data were statistically was analyzed using oneway analysis of variance (ANOVA) according to Tamhane and Dunlop (2000) followed by Duncan's multiple range post hoc test for pairwise comparisons. Differences were considered significant at p < 0.05.

RESULTS AND DISCUSSION
It was clearly evident from Table 1  of moxifloxacin for 21 successive days to albino rats resulting in a significant increase (p<0.05) in the activities of ALT, AST, ALP on 1 st , 7 th , 14 th and 21 st day post treatment beside significant decrease (p<0.05) in the levels of total serum protein and on 1 st , 7 th , and 14 th day post treatment compared with control group. Co-administration of moxifloxacin and Vit. E for 21 successive days to albino rats resulting in a significant decrease (p<0.05) in the activities of ALT, AST, ALP after 1 st , 7 th , 14 th and 21 st day post treatment while the levels of total serum protein and albumin were significantly (p<0.05) increased on the 14 th day post treatment compared with moxifloxacin group. Table 2 illustrated that the administration of moxifloxacin to albino rats resulting a significant (p<0.05) increase in creatinine and urea levels when compared with the control Table 1: The effect of Vit.E (100mg/kg, orally once daily), Moxifloxacin (7.2mg/kg, orally once daily) and their combination for 21 consecutive days on liver enzymes, total proteins, albumin and globulins of rats on 1 st ,7 th ,14 th , and 21 st days of drugs withdrawal (n=5, mean±SE).   It was clearly evident from Table 2 that the administration of moxifloxacin in therapeutic dose for 21 successive days in albino rats resulted in a significant (p<0.05) decrease in CAT, SOD and GPx on1 st , 7 th , 14 th , and 21 st days post treatment when compared with the control group. The coadministration of moxifloxacin with Vit E for 21 successive days to albino rats resulting in significant (p<0.05) increase in CAT, SOD and GPx on 1 st , 7 th , 14 th and 21 st days post treatment when compared with moxifloxacin group. The administration of moxifloxacin for 21 successive days to albino rats resulting in a significant (p<0.05) increase in MDA concentration on 1 st and 7 th , 14 th days post treatment when compared with the control group. While, co-administration of moxifloxacin and Vit E combination for 21 successive days to albino rats resulting in return to nearly normal concentration of MDA when compared with moxifloxacin group.

hiStopatholoGical rESultS
On the 14 th days post treatment Examined sections from liver showed normal hepatic parenchyma with preserved hepatic lobulation. Sections from kidney revealed apparently normal renal structures (Figure 1). Liver sections of rats received vitamin E, showed apparently normal hepatic parenchyma with preserved lobular pattern, cord arrangement, vascular tree, sinusoids, kupffur cells and portal area structures. Kidney sections showed normal nephron units with preserved renal papillae, renal pelvis and stroma ( Figure 2). Liver sections of moxifloxacintreated rats showed multifocal hepatic necrosis of variable sizes, partially replaced by macrophages occasionally with giant cells formation. The hepatic blood vessels were moderately congested and the bile ducts were proliferated with characteristic portal round cells aggregation and portal fibrosis (Figure 3). Examined sections from kidney showed cystic dilatation of few tubules in the medulla and cortex. The renal pelvis revealed focal sloughing and hyperplastic changes in the transitional epithelium. Focal interstitial and perivascular aggregation of round cells and eosinophils were observed. The renal blood vessels were mildly congested with mild perivascular edema (Figure 4). Liver sections of Moxifloxacin + Vit E treated rats showed normal hepatic parenchyma with residual portal biliary proliferation and fibrosis. Minute focal hepatic necrotic areas partially replaced by round cells were also seen. The hepatic blood vessels were mildly congested and the kupffur cells were prominently enlarged and focally proliferated ( Figure 5). Renal Sections of with moxifloxacin + Vit E treated rats showed apparently normal nephron units with mild degenerative changes in some tubular epithelium (cloudy swelling and hydropic degeneration) and cystic dilatation of a few tubules in the cortex and medulla ( Figure 6).   Activities of ALT and AST are acknowledged marker of hepatocellular injury. Raised plasma ALT and AST might be connected with membrane spillage of the hepatocyte cytosolic substance which is reflected in significant rise of the plasma of rodents treated with various dosages of Moxifloxacin (Vahidi-eyrisofla et al., 2015). The administration of moxifloxacin for 21 successive days to rats induced a significant increase in the activities of ALT, AST, and ALP after one day post-administration when compared with control group. The same results were recorded by Ore and Olayinka (2015) who found that an elevation in the activities of serum ALT, AST and ALP beside a significant rise in serum urea in Sprague-Dawley rats of both sexes received oral dose of 8 mg/kg b.w. moxifloxacin for 7 successive days. Co-administration of moxifloxacin and Vit E to mature male rats induce significant decrease in the activities of AST, ALT and ALP in its therapeutic dose at 1 st , 7 th , 14 th and 21 st days compared with group III which treated with moxifloxacin only.  The obtained results in this study revealed that oral administration of moxifloxacin for 21 successive days to mature male rats resulted in a marked decrease of total serum protein, and albumin all over the experimental periods when compared with normal control group. The obtained results were parallel to those obtained by Sadariya et al. (2010) who recorded a significant reduction in serum total protein and serum albumin in male and female wistar rats injected intramuscularly with moxifloxacin at doses of 5 mg/kg repeated at 24 hours interval for 14 days. Coadministration of moxifloxacin + Vit E on 1 st ,7 th , 14 th and 21 st days to mature male rats induces significant increase in total serum proteins and serum albumin when compared with group III which received moxifloxacin only.
The administration of moxifloxacin to albino rats resulted in a significant increase in serum creatinine and urea levels when compared with the control group on 1 st , 7 th , 14 th , and 21 st days post treatment. Also, Ore and Olayinka (2015) who recorded an elevation in serum creatinine and urea in Wistar rats which received oral dose of 1 ml each of moxifloxacin equivalent to 4 mg/kg BW, 8 mg/kg BW, and 16 mg/kg BW, for 7 days.
Antioxidants are substances that when show at low concentrations contrasted to those of an oxidizable substrate (e.g. proteins, lipids, carbohydrates and nucleic acids) altogether delays or suppress oxidation of that substrate (Halliwell et al., 1996). Reactive oxygen species (ROS) are constantly created as a metabolic item by basically all tissues in moderately small amounts. Every mammalian cells contain various distinctive enzymatic and non-enzymatic antioxidants that serve to counteract or limit oxidative tissue damage. The principal line of safeguard against oxidative insult is the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. The non-enzymatic guards incorporate assortment of low molecular weight scavenger, and reductants, and also a few different iron chelators (Halliwell et al., 1994).
An extraordinary supporter of non-enzymatic assurance against lipid peroxidation is Vitamin E (Vit. E), a known free radical scavenger. Vit. E as a lipid solvent, chainbreaking antioxidant plays a critical defensive part against oxidative stress and prevents the creation of lipid peroxides by searching free radicals in biological membranes (Ellis et al., 2017).
It has been suggested that free radicals created after medication by fluoroquinolones, assume a critical part in these antibiotics' toxicity (Thompson, 2007). Numerous antioxidant enzymes, including SOD, CAT, and glutathione GPx, are viable in evacuating destructive ROS. Lacking action of intracellular antioxidant enzymes can make damage to cell structures. Whenever unbalanced, it might prompt oxidation of polyunsaturated fatty acids in lipids, amino acids in proteins, and obliterate DNA (Mari et al., 2010;Beberok et al., 2015). In this study, it has been observed that moxifloxacin causes critical decrease in the activities of the antioxidant enzymes: SOD, CAT, and GPx in melanocytes. SOD ensures cells by dismutating superoxide anion into the proradical hydrogen peroxide, which thusly is inactivated to water and oxygen by catalase or other H 2 O 2 -evacuating enzymes such as glutathione peroxidase (Finaud et al., 2006).
Vitamin E is a lipid-solvent vitamin, of which α-tocopherol is the most strong. Vitamin E acts as an antioxidant in cells, intruding on the spread of lipid peroxidation in the plasma membrane and in this way safeguarding membrane integrity (Chow, 1991).Very responsive molecules called free radicals can cause tissue damage by responding with polyunsaturated fatty acids in cell membranes, the degree of tissue damage is the consequence of the harmony between the free radicals created and the antioxidant protective defense system (Lawrence and Adrianne, 1987).Vitamin E is the best lipid-dissolvable, chain-breaking antioxidant, shielding cell membrane from peroxidative damage. Free radicals have been involved in the improvement of degenerative sicknesses and conditions (Lester, 1991). When the antioxidant defense in the human body becomes overwhelmed, oxidative stress to the parts frequently happens, initiating inflammatory, adaptive, and reparative procedures (Borut and Rok, 2014). As of late, Vit E is being widely examined because of its activity against oxidative stress. Its defensive part on biological membranes is identified with its impact on delaying the side effects of aging (Enesco et al., 1980). The in vivo function of Vit E as an antioxidant has not yet been completely studied (Kumar and Adarad, 1988). Ongoing investigations have uncovered that Vit. E has an antioxidative activity in shielding cells from damage by highly responsive superoxide free radicals (Mokhtari et al., 2017).
Vitamin E normalized levels of catalase, superoxide dismutase, glutathione peroxidase, malondialdehyde and enhanced histopathological changes occur in liver and kidney induced by receiving of moxifloxacin. The possible pathway can be clarified through structure of Vitamin E, the side chain in the 2-position encourages the consolidation and maintenance of Vit. E in bio films, with the goal that the 6-position is responsible for rummaging free radicals and ending lipid peroxidation. Antioxidant impact of vitamin E is shown through insurance of polyunsaturated fatty acids from oxidation by reactive oxygen species making adjustment of membrane and breaking of antioxidant chains that counteract responsive oxygen species harm to membrane.
In this study Vit. E utilized in a dose of (100 mg/kg, orally once daily) to clarify the hepatic-nephroprotective effect on rats, we find a critical rise in the activities of anti-oxidative