In the modern society, our dependency on ready to eat food increases due to lifestyle we have. These processed foods are very harmful for mankind upon their consumption and they could even adversely affect the quality of food [16]. AA is one of those harmful processed chemicals. AA is formed when carbohydrate rich food is processed at high temperature (> 120 ◦c). There are other modes of exposure, from where AA can enter in human body such as its use in industrial products like water purification, cosmetics, laboratories and even present in cigarette smoke. Its presence in coffee, green tea and confectionaries had also been detected [1]. Hence, toxicity caused due to AA is one of the major health problems.
AA is electrophilic in nature and when it enters in body, it can react with nucleophilic compounds containing SH, NH2 and OH groups and form Hb-AA adducts. AA is a small organic molecule, hence is readily absorbed and thereafter distribute to all the tissues in a rapid manner. AA get converted to its genotoxic form, glycidamide by epoxidation reaction through CYP2E1 enzyme system which are aniline hydroxylase and amidopyrine-N-demethylase. Glycidamide is more reactive toward protein and form both GA-Hb and GA-DNA adducts [17]. During AA metabolism it get conjugated with GSH and form GSH adduct, which rapidly transformed to mercapturic acid and eliminated out from the body. Almost every organ of the body is a target of AA toxicity. Long term AA exposure can cause paresthesia, muscle weakness, muscular atrophy, absence of tendon reflex, hind limb splaying, tremor, illusion or cognitive dysfunction [2].
Use of plant product as medicinal purpose had always gain interest of researchers because of their high bioavailability and non- toxic nature. There are increasing evident that most of the disorders results from oxidative imbalance caused by ROS. To prevent the oxidative stress generated by toxic chemicals, antioxidant role of bioactive phytochemicals has been reported.
To reduce the risk of AA toxicity phytochemicals like phenolic acids are used. CA (3,4-dihydroxy cinnamic acid) a plant flavonoid produced through the secondary metabolism, is most abundant bioactive compound. The radical scavenging properties associated with the structure of CA defend against oxidative stress preventing the production of ROS (reactive oxygen species) and in doing so it has many other biological properties such as anti- inflammatory, antimutagenic, cardioprotective, reno-protective, reduce blood sugar level as well as prevent cancer and slow the aging processes in cells responsible for degenerative diseases [18].
Result of this study shows that in comparison with control there is a significant reduction in the percentage of hemoglobin upon AA intoxication. Hemoglobin is dependent on red blood cells population and the number of red blood cells had also been decrease significantly. This may be due to hindrance in the synthesis of hemoglobin or hemoglobin destruction. Activity of ALAD had also significantly decreased in AA administered rats. ALAD is -SH containing enzyme involved in the synthesis of heme [19], AA had high affinity to conjugate with -SH group. This leads to decrease in the activity of ALAD and ultimately results in decrease hemoglobin content, which leads to anemia. Its inactivation also induces the accumulation of ALAS, which accelerate the production of ROS and ultimately induce oxidative stress.
Sub-chronic exposure of AA accelerates ROS generation process which disturbed the oxidant/ antioxidant level in the cell. This resulted into decline activity of antioxidant enzymes (GR, GPx, GST, G-6-PDH) in liver, kidney and brain tissue. This may be due to cellular injury or death of healthy cells, which are responsible to counter the oxidative stress. Therapy of CA maintained antioxidant enzymes toward normal due to its radical scavenging property.
Lipid peroxidation is the metabolic process in which ROS results in peroxidation of membranous lipids. Our study demonstrated that the level of TBARS significantly increased in the AA intoxicated rats, might be because of the failure in detoxification of AA. Treatment with CA subsequently reduced the level of LPO toward normal. This may be because of antioxidant nature of CA, it is characterized by the presence of a benzene ring, a carboxylic acid group and hydroxyl groups.
Aniline hydroxylase (AH) and aminopyridine-N-demethylase (AND) are CYP 450 enzymes of CYP-450 enzymatic system and responsible for drug metabolism. Activities of these enzymes was determined in liver microsomes, originated from smooth endoplasmic reticulum (SER), a major site of lipid biosynthesis. In this present study activities of these drug metabolizing enzymes were significantly declined in AA intoxicated rats, suggesting SER damage. Therapy of CA restored the activities of these enzymes toward control.
In the present study, elevated level of AST, ALT, ALP, GGT, LDH, albumin and bilirubin in serum has been recorded after sub-chronic exposure of AA. These enzymes are cytoplasmic in origin and may release into the circulation due to the damage in cell membrane integrity or due to exposure of toxicant and are sign of liver damage induced by AA toxicity. This increase activity of serum enzymes may be due to the bipolar nature of AA and it can conjugate with -SH and α-NH2 group of amino acids. Treatment of animals with CA showed protection against AA induced hepatic damage. CA treated animals showed the restored activity of AST, ALT, ALP, GGT, LDH, albumin and bilirubin which may be due to its ability to repair liver damage and maintain the integrity of plasma membrane. Increased bilirubin is due to increase in the production of bilirubin more than the normal or due to damage or blockage of excretory duct of liver. Our results are in agreement with the result of Uthra et al. 2022 [20]. Elevated level of cholesterol and triglycerides in AA administered group also demonstrate the liver damage. These results indicated that changes in plasma lipoproteins can serve as sensitive and simple markers for rat’s liver disorders caused by AA. Histopathological data of this study clearly signify the liver damage characterized by development of cytoplasmic vacuolation, hepatocyte necrosis and lymphocytic infiltration upon AA intoxication [21]. Treatment of CA help in the restoration of altered parameter toward normal values.
Level of serum urea, uric acid and creatinine, main clinical parameters for assessing renal damage altered upon AA administration. Level of urea, uric acid and creatinine may be elevated because AA intoxication can impair brush border epithelium and make them impermeable to urea, uric acid and creatinine [22], or it may be due to excessive production or degradation of purine and pyrimidine. Treatment with CA restored the altered parameters toward normal. It could be possible by the antioxidant property of CA which ultimately modulates the key enzymes and recover the renal damage, which is also evident by our histological analysis of kidney.
Neurotoxicity of AA was reported by many researchers [23]. These researchers reported that AA produces central and peripheral distal axonopathy. AA exposure also leads to decrease the level of norepinephrine in the hippocampus and noradrenergic axons in the prefrontal cortex. This study demonstrate that AA intoxication caused inhibition in the activity of AChE and BuChE in brain. Administration of CA significantly recouped the AChE and BuChE activity towards normal highlighting its neuroprotective property. After the therapy of CA, improvement in the histoarchitecture of brain tissue also supported these results.
Genotoxicity of AA is associated with its biotransformation to a highly reactive glycidamide. Glycidamide can directly conjugate with DNA and form glycidamide-DNA adducts [24]. Genotoxic assessment by comet assay shows a significant increase in the tail length and tail DNA in the liver, kidney and brain tissue of AA administered rats. CA has an anti-genotoxic effect, due to its antioxidant property. The antioxidant property of caffeic acid is due to the presence of -OH group in its structure. Not only the OH group present on para position makes it a potent antioxidant but, the presence of second hydroxyl group in the ortho position increases its antioxidant property due to an additional resonance stabilization and form o-quinone.