Hepatotoxicity Implies Chemical-Driven Liver Damage Induced By Certain Medicinal and Other Chemical Agents


Mohamad Sayyed Bakheet , Haredy Hassan *

Citation: NA

Copyright NA


Abstract:

There are increasing evidences that free radicals and reactive oxygen species play a crucial role in the various steps that initiate and regulate the progression of liver diseases. Oxidative stress in hepato-toxicity resulting from hepato-toxicity resulting from hepato-toxicity resulting from hepato-toxicity resulting from increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses actively contributes to excessive tissue remodeling. Drug-induced nephropathy is reported to be the third most common cause of acute renal failure in hospitalized patients. Excess ROS production and depressed antioxidant increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses actively contributes to excessive tissue remodeling. Drug-induced nephropathy is reported to be the third most common cause of acute renal failure in hospitalized patients. Excess ROS production and depressed antioxidant increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses actively contributes to excessive tissue remodeling. Drug-induced nephropathy is reported to be the third most common cause of acute renal failure in hospitalized patients. Excess ROS production and depressed antioxidant increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses actively contributes to excessive tissue remodeling. Drug-induced nephropathy is reported to be the third most common cause of acute renal failure in hospitalized patients. Excess ROS production and depressed antioxidant hepato-toxicity resulting from hepato-toxicity resulting from hepato-toxicity resulting from increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses actively contributes to excessive tissue remodeling. Drug-induced nephropathy is reported to be the third most common cause of acute renal failure in hospitalized patients. Excess ROS production and depressed antioxidant increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses actively contributes to excessive tissue remodeling. Drug-induced nephropathy is reported to be the third most common cause of acute renal failure in hospitalized patients. Excess ROS production and depressed antioxidant hepato-toxicity resulting from hepato-toxicity resulting from hepato-toxicity resulting from hepato-toxicity resulting from increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses actively contributes to excessive tissue remodeling. Drug-induced nephropathy is reported to be the third most common cause of acute renal failure in hospitalized patients. Excess ROS production and depressed antioxidant increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses actively contributes to excessive tissue remodeling. Drug-induced nephropathy is reported to be the third most common cause of acute renal failure in hospitalized patients. Excess ROS production and depressed antioxidant defence mechanism are responsible for nephrotoxicity.So, pharmacological studies in this work were done to evaluate: presence of protective effects of an antioxidant Hesperedine on carbon tetrachlorideinduced hepatic toxicity and nephro-toxicity, to evaluate its effects on oxidants and antioxidants parameters and to evaluate its effect on kidney and liver functions and histo-pathological changes.Liver enzymes level AST and ALT : was increased significantly in rats treated with CCl4 but decreased significantly in rats treated with antioxidant HDN (100 mg/ kg/ day) and in rats treated with antioxidant HDN (200 mg/ kg/ day) . in comparison between antioxidant treated rats groups liver enzymes level was decreased significantly in rats treated with antioxidant HDN (200 mg/ kg/ day) than in rats treated with antioxidant HDN (100 mg/ kg/ day) .Serum creatinine level: was increased insignificantly in rats treated with CCl4 but decreased insignificantly in rats treated with antioxidant HDN (100 mg/ kg/ day) and in rats treated with antioxidant HDN (200 mg/ kg/ day). in histo-pathological changes.Liver enzymes level AST and ALT : was increased significantly in rats treated with CCl4 but decreased significantly in rats treated with antioxidant HDN (100 mg/ kg/ day) and in rats treated with antioxidant HDN (200 mg/ kg/ day) . in comparison between antioxidant treated rats groups liver enzymes level was decreased significantly in rats treated with antioxidant HDN (200 mg/ kg/ day) than in rats treated with antioxidant HDN (100 mg/ kg/ day) .Serum creatinine level: was increased insignificantly in rats treated with CCl4 but decreased insignificantly in rats treated with antioxidant HDN (100 mg/ kg/ day) and in rats treated with antioxidant HDN (200 mg/ kg/ day). in comparison between antioxidant treated rats sed insignificantly in rats treated with CCl4 but decreased insignificantly in rats treated with antioxidant HDN (100 mg/ kg/ day) and in rats treated with antioxidant HDN (200 mg/ kg/ day). in comparison between antioxidant treated rats comparison between antioxidant treated rats groups liver enzymes level was decreased insignificantly in rats treated with antioxidant HDN (200 mg/ kg/ day) than in rats treated with antioxidant HDN (100 mg/ kg/ day) . So, we recommend uses of antioxidant Hesperedine as it has a valuable role in improvement of liver functions and as a prophylactic of hepatic and renal tissues against toxicity achieved by free radicals. 

Description:

I. INTRODUCTION Hepato-toxicity Implies chemical-driven liver damage. Certain medicinal agents, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the organ. Other chemical agents, such as those used in laboratories and industries, natural chemicals (e.g., microcystins) and herbal remedies can also induce hepatotoxicity. Chemicals that cause liver injury are called hepatotoxins. Chemicals often cause subclinical injury to liver which manifests only as abnormal liver enzyme tests. Drug-induced liver injury is responsible for 5% of all hospital admissions and 50% of all acute liver failures. More than 75 % of cases of idiosyncratic drug reactions result in liver transplantation or death (Ostapowicz et al., 2002; McNally and Peter, 2006).

Mitochondria are prominent targets for the hepato-toxicity of many drugs. Dysfunction of these vital cell organelles results in impairment of energy metabolism and an intracellular oxidant stress with excessive formation of reactive oxygen species and peroxy-nitrite. Induction of cytochrome P450 isoenzymes such as CYP2E1 also promotes oxidant stress and cell injury, once hepatocellular function is impaired, accumulation of bile acids causes additional stress and cytotoxicity. Cell injury, gut-derived endotoxin or a combination of both also activate Kupffer cells and recruit neutrophils into the liver. Although responsible for removal of cell debris and part of the host-defense system, under certain circumstances these inflammatory cells initiate additional liver injury (Jaeschke et al., 2002).

Drug-induced liver diseases mimic all forms of acute and chronic hepatobiliary diseases. However, the predominant clinical presentation resembles acute icteric hepatitis or cholestatic liver disease. The former is the more serious and often has a 10% mortality rate, regardless of the causative drug, (Zimmerman, 1999; and Kaplowitz, 2002).

Acute icteric hepatitis is accompanied by markedly elevated serum transaminase levels and a minimal increase in the level of alkaline phosphatase. Coagulopathy and encephalopathy are present in more severe cases. Cholestatic disease (which is also referred to as cholestatic hepatitis) is not usually life threatening; it presents with jaundice, pruritus, and marked increases in alkaline phosphatase levels, as well as mild increases in alanine aminotransferase (ALT) levels. Mixed injury patterns with intermediate to marked increases in ALT and alkaline phosphatase levels can resemble atypical hepatitis or granulomatus hepatitis, (Kaplowitz, 2002).

Biochemical markers (e.g. alanine transferase, alkaline phosphatase and bilirubin) are often used to indicate liver damage. Liver injury is defined as a rise in either (a) ALT level more than three times of upper limit of normal (ULN), (b) ALP level more than twice ULN, or (c) total bilirubin level more than twice ULN when associated with increased ALT or ALP, (Bénichou, 1990 and Mumoli et al., 2006). 

Oxidative stress in hepatotoxicity, resulting from increased generation of reactive oxygen species (ROS) and other reactive intermediates as well as by decreased efficiency of antioxidant defenses, actively contributes to excessive tissue remodeling, (Ismail and Pinzani ,2009). 

Indeed, oxidative stress, presumably by favoring mitochondrial permeability transition, is able to promote hepatocyte death (necrotic and/or apoptotic). In some of clinically relevant conditions, generation of ROS within hepatocytes may represent a consequence of an altered metabolic state (like in NAFLD and NASH) or of ethanol metabolism (as in ASH), with ROS being generated mainly by mitochondrial electron transport chain or through the involvement of selected cytochrome P450 isoforms like cytochrome P2E1 (CYP2E1), (Tilg and Hotamisligil ,2006). 

Glutathione (GSH) is a critical cellular antioxidant. After GSH depletion with buthionine sulfoximine (BSO), the toxicity of ethanol, iron, arachidonic acid, and acetaminophen was strikingly enhanced, (Chen et al., 1997; Chen and Cederbaum, 1998; Sakurai and Cederbaum, 1998; Wu and Cederbaum, 1999). 

Cytochrome P4502E1 (CYP2E1), the ethanol-inducible form, metabolizes and activates many toxicologically important substrates, including ethanol, carbon tetrachloride, acetaminophen, and N-nitrosodimethylamine, to more toxic products, (Guengerich et al. ,1990; Koop ,1992). CYP2E1- dependent ethanol metabolism produces oxidative stress through generation of reactive oxygen species (ROS), a possible mechanism by which ethanol is hepatotoxic, (Dianzani , 1985 and Bondy, 1992). Induction of cytochrome P4502E1 by ethanol is a central pathway by which ethanol generates oxidative stress, and in the intragastric model of ethanol feeding a prominent induction of CYP2E1 occurs along with significant alcohol liver injury, (Morimoto et al. ,1994; Nanji et al. , 1994). 

Immunochemical studies indicate that the cellular site of covalent binding correlates with the toxicity, (Roberts et al., 1991 and Hart et al., 1995). Recent work shows that nitrated tyrosine occurs in hepatic centrilobular cells. These adducts colocalize in cells containing the acetaminophen-protein adducts, (Hinson et al., 2000). Peroxynitrite, a highly reactive nitrating and oxidizing species formed by the rapid reaction of nitric oxide (NO) and superoxide, produces nitrated tyrosine, (Pryor and Squadrito, 1995; Beckman, 1996).

Carbon tetrachloride is a colourless liquid, non flammable, and is heavier than air, (Etim et al., 2008). Consequently, it has been widely used as a fire extinguisher being useful for fighting fires near electrical equipment because it does not conduct electricity, (The World Book Encyclopedia, 1992). Carbon tetrachloride is very toxic and because of this, most of its uses in households and industries have been suspended, (Etim et al., 2008). Consequently, little is known about the early effects of this organic solvent in vivo, particularly on mitochondrial function. It has been shown recently in a murine model of liver fibrosis that chronic administration of CCl4 for 6 weeks led to mitochondrial DNA (mtDNA) alterations, reduced glutathione (GSH) depletion and decreased aconitase activity (Mitchell et al, 2009), overexpression of Bcl-2 reduced liver fibrosis for the first 3 weeks of treatment by protecting hepatocytes against mitochondrial damage, but subsequently failed to prevent fibrosis with the persistence of the aggression. CCl4 is activated by cytochrome P450 (CYP) 2E1, and very marginally by other CYPs (CYP2B and CYP3A), to form the trichloromethyl (CCl3. ) free radical, which can react with oxygen to produce the trichloromethyl peroxy radical (CCl3OO. ). Both radicals are highly reactive species that may covalently bind to macromolecules to form nucleic acid, protein and lipid adducts. However, the evidence for such interactions with liver DNA in vivo is limited, (Recknagel et al., 1989 and Weber et al., 2003).

In this study, we used an in vivo model to explore the very early toxic events, particularly regarding mitochondria, occurring after CCl4 administration. Inhibition of CCl4 activation by the CYP2E1 inhibitor diethyldithiocarbamate (DDTC) and impairment of CCl4-induced lipid peroxidation by antioxidants allowed us to establish a direct link between lipid peroxidation and mitochondrial alterations. Antibiotics, commonly used aminoglycosides, are nephrotoxic agents. Their nephrotoxicity is mainly attributed to induction of OS and depletion of antioxidat enzyme activities in kidney. Inducible nitric oxide synthase, nuclear factor kappa-B, nitogen-activated protein kinase (iNOS/NFκB/p38MAPK respectively) pathway, OS taking place in this axis, is involved in gentamicin-induced nephrotoxicity, (Tugcu et al. ,2006 and Ozbek et al. ,2009). The protective effect of anti oxidants and reactive oxygen scavenger agents against gentamicin-induced nephrotoxicity. Antineoplastic agents are commonly used for the treatment of metastatic cancers. Some of these are nephrotoxic, (Ozbek et al., 2010 and Maniu et al., 2011).Excess ROS production and depressed antioxidant defence mechanism are responsible for nephrotoxicity. Cisplatin is the well-known and commonly used antineoplastic and nephrotoxic agent. Other nephrotoxic anticancer agents are carboplatin, methotrexate, doxorubicin, cyclosporine, and adriamycin. Immunosuppressant such as sirolimus and cyclosporine leads to nephrotoxicity via OS, (Giustarini et al., 2009).

In this era, analgesics, especially paracetamol and acetaminophen (APAP), and nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used throught the world. Paracetamol and APAP are nephrotoxic drugs. Several in vitro and in vivo studies showed that analgesics nephrotoxicity is caused by increased ROS in kidney, (Zhao et al, 2011) showed the increased ROS, nitric oxide, and MDA levels, together with depleted glutathione (GSH) concentration in the kidney of rats. However, rhein, Chinese herb, can attenuate APAP-induced nephrotoxicity in a dose-dependent manner, (Zhao et al., 2011).Some studies showed a significant increase in MDA and decreases in GSHPx, CAT, and SOD activities in APAPtreated rat kidneys. These findings support the induction of OS in rat kidney by APAP. Significant beneficial changes were noted in serum and tissue OS indicators in rats treated with strong antioxidant pineal hormone melatonin and curcumin, ( Ilbey et al. ,2009 ; Cekmen et al. ,2009) ,reported increased OS and TNFalpha production in rat tissues, (Ghosh et al. ,2010) ,reported that diclofenac (NSAID) leads to nephrotoxicity by increasing intrarenal ROS in rat kidney, and antioxidant, Nacetylcysteine, prevents kidney damage, (Efrati et al. ,2007). GSH is able to regenerate the most important antioxidants, Vitamins C and E, back to their active forms; it can reduce tocopherol radical of Vitamin E directly, or indirectly, via reduction of semidehydroascorbate to ascorbate. The capacity of glutathione to regenerate the most important antioxidants is linked with the redox state of the glutathione disulphide-glutathione couple (GSSG/2GSH), (Pastore et al. , 2003). Hesperidin is a flavanone glycoside named after the term `Hesperidium`, referring to citrus fruits which are the main source of hesperidin. Hesperidin and its aglycone are common dietary flavonoids due to being large compounds of citrus fruits (alongside naringenin) and especially the peels and pericarp, (Kanes et al, 1993). 

There are inhibitory effects of hesperitin on two intestinal transporters, the OATP2B1 (Organic Acid Transporting Polypeptide 2B1) transporter and MRP2 (Multidrug Resistance Protein 2). OATP2B1 appears to be acutely inhibited with supplementation of hesperidin, whereas low doses of hesperidin over a few weeks appear to downregulate the MRP2 transporter .It is notable to know that the OATPs play a fundamental role in the transport of drugs across the cell membrane, particularly in the liver and kidney. In the liver, OATPs are expressed on the basolateral membrane of hepatocytes, transporting compounds into the hepatocyte for biotransformation (Price et al, 2006).

A 0.079% hesperidin suspension given to rats for eight weeks is able to increase the overall exposure (147%) and peak concentration (138%) to the drug pravastatin, (Shirasaka et al ., 2013) which is thought to be due to inhibition of the transport protein known as Multi-drug Resistance Protein 2 (MRP2) that mediate pravastatin efflux into the intestines after absorption, (Tamai , 2012) . There appear to be antioxidant effects in the brain where hesperidin reduces the increase in lipid peroxidation during cognitive damange, but this appears to be indirect through nitric oxide signalling (inhibition) rather than a direct antioxidant effect, (Olivenza et al. , 2000; McEwen, 2001; Alexaki et al., 2004 and Takeda et al., 2008). Damage of DNA is reduced by hesperidin (Sahu et al. ,2013).Hesperidin intake in diabetic rats appears to significantly but not fully reduce levels of the (vascular endothelial growth factors )VEGF and PKCβ (Protein kinase cβ), and it is thought that the reduction in signalling (from VEGF towards PKCβ) causes a protective effect on the retinal membrane and reduces the progression of diabetic retinopathy, (Donnelly et. al , 2004 ; Liu et al. , 2008 ; Wang et al. , 2010 and Kumar et al. , 2012). 

II. MATERIALS AND METHODS

This study was conducted on Thirty two male albino rats. Animals were obtained from the animal house of faculty of medicine, Al-Azhar University. Their weight ranged between 160-200 grams each at the beginning of the experiment. Rats were housed in four groups with 8 rats each in clean capacious macrolane cages under standard laboratory conditions, including good aerated room with suitable temperature (25±5°C), maintained at good light, standard rodent food and water were available. 

CCL4: El-Naser Pharmaceuticals chemical company, Egypt

Hesperidine (HDN): Sigma, Aldrich.

-Saline, El-Naser Pharmaceuticals chemical company, Egypt.

- Phosphate buffered saline, Hi-media- Lab. Pvt. Inc., USA.

-SOD kit: Biochemical Enterprise, Italy

-Malon-Di-Aldehyde: Biochemical Enterprise, Italy

-Glutathione reduced determination kit: Biochemical Enterprise, Italy

- ALT and AST determination kits: Centronic_Gmbh, Germany.

-Serum Creatinine determination kits: Diamond., USA. 

In the present study, the animals were divided into the following groups. Each group consisted of 8 rats:

Group I : These animals received a vehicle for HDN (i.e. CarboxyMethylCellulose) by oral route for eight days and on 8 th day, they were administered the subcutaneous injection of olive oil (.Tirkey)2005 ,

Group II: These animals received vehicle for 10 days and were challenged with CCl4 2 ml/kg/s.c. (40% v/v in olive oil) on 8th day (Mandal and Sinha, 2002)

Group III: These rats received only HDN 100 mg/kg/p.o. daily for 10 days CCl4+ HDN (100): Rats received HDN continuously for 8 days. On eight day just after HDN treatment they received CCl4 2ml/kg/s.c in olive oil. HDN was further continued for 2 more days. (Tirkey, 2005)

Group IV: These rats received only HDN 200 mg/kg/p.o. daily for 10 days CCl4+ HDN (200): Rats received HDN continuously for 8 days. On eight day just after HDN treatment they received CCl4 2ml/kg/s.c in olive oil. HDN was further continued for 2 more days.( ( Tirkey, 2005) 

Forty-eight hours after the last CCl4 injection, rats were sacrificed and blood samples were collected, centrifuged and the serum from each animal was kept in epindorff tubes in the deep freezer at (-20°C) until analyzed for liver functions.

After animals were sacrificed livers were immediately excised, rinsed from blood in ice cold saline, blotted dry by filter papers. Small piece of each liver was fixed in 10% phosphate-buffered formalin for histological examination. About 0.5 gm of each liver was homogenized by ultra sonic homogenizer in 5ml ice-cold phosphate bufferd saline (PBS) to obtain ultimately10% (w/v) whole liver homogenate (Ezz et al., 2011 ;Fahmy and Hamdi, 2011). The homogenate was centrifuged at 3000 rpm for 15 min and the resultant supernatant was stored at -20°C until used for determination of reduced glutathione (GSH), malondialdhyde (MDA), superoxide dismutase (SOD) and hydroxyproline concentration.

Determination of liver function:

Determination of alanine aminotransferase (ALT) (IU/L): (Thomas , 1998)

Determination of aspartate aminotransferase (AST) (IU/L): (Thomas , 1998).

Determination of kidney function:

Determination of Serum Creatinine (mg/dL): (Murray, 1984)

Determination of hepatic reduced glutathione mg/g tissue: (Beutler, 1963).

Determination of hepatic superoxide dismutase U/g tissue: Nishikimi et al )1972 ,.

Determination of hepatic lipid peroxide (malondialdehyde) nmol/g tissue: (Satoh, 1978)

III. RESULTS

                            
                           
                                  
Normal liver tissue
-Normal architecture –normal rows
-No inflammatory cell infilterate
-Normal cellular appearance
-Normal apparent nuclei

                        
-Extensive damage.
-Very sever vaculation
-Inflammatory cell infilteration
-Disruption of the lattice nature of hepatocytes and damaged hepatocyte cell membrane
-Irregular architecture (damaged sinusoids, rows and disintegrated central vein)
-Degenerated nuclei

                    
-Presence of vaculation but less than control positive group.
-More eosinophis infiltration than control positive group.
-Better viability and less damage than control positive group.
-Nuclei are healthier than control positive group.
- Less disruption of the lattice nature of hepatocytes and less damaged hepatocyte cell membran
- More regular architecture and rows than control positive.

                             
-Faded vaculation (very mild)
-Architecture and rows are so close to normal.
-Normal viability
-Less infiltration by the inflammatory cells than treated groups by (HDN100)
-Normal nuclei and cell membranes
-Normal central vein and sinusoids.