Turmeric Plays Protective Role against Paraben Induced Hepatic and Renal Lesions in Albino Mice

| Preservatives have been used since prehistoric times and today is a recurring topic in world debate. Paraben is among the top class preservatives used in food, pharmaceutical agents, drinks, beautifying agents and numerous different items. So purpose of study was to determine the possible hepatic and nephrotoxic effects of Butyl paraben in mice and to explore the possible curative and protective impacts of turmeric. Fifty mice randomly divided in five groups, i.e. Control, dose group (BP), dose and antidote group (BP+AD), vehicle control group (VC) and antidote group (AD). A sub lethal concentration containing 15ug/g B.W of BP was given with and without turmeric orally, dissolved in corn oil, used as vehicle too. Dissections were made on 31st day, twenty-four hours after the last dose administration. Results showed that continuous administration of butyl paraben (BP) had decreased the organs weight as compared to control. Regarding biochemical parameters results had shown an increased enzymatic level of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and a significant increase in bilirubin levels in BP group. Significant raised patterns were recorded for urea and creatinine in BP intoxicated mice against control. Histological defects observed were necrosis, pyknosis, increased sinosuidal spaces, portal hypertension, glomerulosclerosis, abnormal vascular pole in liver and kidney respectively. But when turmeric powder was coadministered with BP, these damages were reversed to a great extent. So, this study has proved the protective role of turmeric against toxicity induced by BP in female albino mice. Novelty Statement | Curcumin, an active component of turmeric along with its proven anti-fungal and antimicrobial activities also detoxify Paraben (suspected car-cinogen and endocrine disruptor) instigated damages. Article History Received: October 05, 2019 Revised: December 17, 2019 Accepted: January 08, 2020 Published: January 10, 2020 Authors’ Contributions CA and Asmatullah conceied, executed and supervised the study. CA wrote the manuscript. ZR and Memoona conducted the experiments. SA did histopathology and slide labelling.


Introduction
F rom previous few years, concern has aroused regarding possible unfriendly impacts of chemical additives on hepatic and renal functions. Parabens are much highlighted name of commercial industry as they are used in daily used products of humans and we all humans are exposed to this compound in one way or the other (e.g. food products, are excreted out in bile and urine (Nowak et al., 2018). Toxicity and effectiveness of parabens mainly hang on the length of alkyl chain substituent. Greater the length of alkyl chain of substituent greater the toxicity of paraben so the order is as follows: methyl paraben < ethyl paraben < butyl paraben < propyl paraben and so on. It is a very common constituent of more than 90 percent cosmetic and food products where it is mainly used as preservative (Zhang et al., 2017).
Humans are primarily exposed to this compound via processed food like jams, jellies, canned fruits and beverages etc. (Boberg et al., 2016). Dermal application of cosmetic products like lipsticks, eye shadows, perfumes, foundations etc. are also one of main sources of absorption of this compound in human body. As females are more exposed towards cosmetic products so their exposure to this compound is greater in comparison to males (Giulivo et al., 2016). Massive consumption magnifies the BP level with respect to concentration subjected to time. After absorption by skin or oral ingestion BP partly breaks down into PHBA and alcohol. Some intestinal enzyme also participates in this process.
Liver is the main detoxification center of body, it plays an important role in production of anti-oxidants and maintains the metabolic mechanism of body. Raised production levels of AST, ALP and ALT are critical indicators of liver abnormality caused due to any exogenous or endogenous factor like toxic compound or virus etc. (Abbas et al., 2010;Boberg et al., 2010). BP is known to cause damaging effects on oxidizing organs like liver and kidney. BP may cause increased number of free radicles in hepatic cells and may lower the concentration of glutathione, superoxide dismutase and ascorbic acid (Watkins et al., 2015).
Spices and herbs are one of the most primitive therapeutic agents known to mankind. Curcuma longa (turmeric) is one of the cheap and easily available spice. Curcumin is an active and most important component present inside turmeric. It has many anti-oxidative and anti-carcinogenic characters (Nabavi et al., 2012;Embuscado et al., 2015;Shi et al., 2019). The basic working mechanism of turmeric is to increase the amount of anti-oxidants inside body, it also decreases the amount of reactive oxygen species thus it can lower the oxidative stress efficiently as also reported by (Ahmida, 2012;Shi et al., 2019). Other scientific researchers i.e. El-Mahalaway (2015) have also reported turmeric as an effective antidote against the induced toxicity of poisonous compounds. Curcumin is the best ingredient of turmeric that naturally boosts up immunity and has anti-microbial and antifungal properties.
The main research goal of this study is to indicate all the possible toxic and damaging effects of BP on liver and kidney at 15µg/g B.W. Turmeric was used as an antidote to investigate its therapeutic potential against the probable induced toxicity by butyl paraben. As our work was on mammalian model so results can be extrapolated to human beings.

Experimental animals
Research was carried on Swiss Webster mice (Mus musculus), reared in the animal house of Zoology Department, University of the Punjab, Lahore, Pakistan. Light cycle was kept constant i.e. 14/10h light/dark cycle. Temperature 27±2 ºC was maintained for the experiment with 40-50% factor of humidity. Readymade chick feed pellets were used (Feed No 10, National feeds, Lahore, Pakistan). Healthy growth of mice was ensured by adding supplements during husbandry. Feed and water were given ad libitum. Randomly selected one month old (weighing 10-15g) animals were placed in five groups. Each group contained 10 mice. The study was properly done in keeping the view of ethical guidance of local committee of University of the Punjab, Lahore.

Chemicals
N-butyl paraben of analytical grade was purchased from Sigma Aldrich, Martin Dow Marker Specialties (Pvt.) Ltd D-7, Shaheed-e-Millat Road, Karachi-75350, Pakistan. Raw turmeric was purchased from local market, washed, dried and grinded to get raw turmeric powder.

Experimental design
50 randomly selected prepubertal mice were divided in 5 groups and placed in separate cages. Group I, control animals were maintained without any treatment. Animals of group II designated as BP, was orally exposed to BP 0.1 ml solution having desire amount of dose (15µg/g B.W). 0.1 ml of BP solution along with 0.1 ml of turmeric solution was given to group III (BP+AD). 0.1ml of corn oil was orally administered to vehicle control (VC) group IV. While group V (AD) only administrated with turmeric solution (0.04mg/0.1ml) (Hawlings and Kalma, 2017). Mice were treated with single dose daily for 30 days. After completion of treatment, animals were sacrificed following procedures and guidelines by Ethical committee on animal care and experimentation of University of the Punjab, Lahore, Pakistan. Required viscera were dissected out and blood extracted through cardiac puncture under deep chloroform anaesthesia.

Determination of biochemical parameters
10ml serum collection tubes were used to collect blood sample and were maintained at room temperature for 30 min. Samples were centrifuged for 10 min to obtain serum at 4 ºC. Bayer's kits were used according to the prescribed protocols by the manufacturer. Total proteins, bilirubin, aspartate transaminase, alanine transaminase, alkaline phosphatase, creatinine and urea concentrations be assayed.

Morphology and morphometric observations
The livers and kidneys of dissected mice of all groups were examined. Morphological analysis was done by using Labomed microscope USA. Their physical appearance and shape was noticed. For morphometric observations kidneys and livers were weighed on digital balance. Width and length of liver and kidney was measured accurately with the help of digital Vernier calipers.

Histopathology of kidney and liver tissues
For histopathology, livers and kidneys were kept in formalin and then tissues were transferred to series of alcoholic concentrations i.e. 70%, 80%, 90% and then 100% for dehydration. For the clearance of tissues for paraffin embedding, xylene was used. Embedding of tissues was done in liquid paraffin wax. Small thin sections of 5 microns were cut by rotatory microtome. Tissue sections having paraffin wax were transferred to a glass slide and dried at 40 ºC. For Staining wax was removed by treating with xylene. Followed by rehydration, sequence of alcohol washes was reversed i.e. from higher to lower grade. Hematoxylin and eosin were used for staining nucleus and cytoplasm respectively and slides were viewed under light microscope, SWIFT MD3500. High mega pixel camera was used for taking microphotographs

Statistical analysis
Data was analyzed using SPSS version 21. Means ± SEM was recorded. Analysis of variance (ANOVA) was used to determine the mean differences between various groups. A difference between the groups was considered at p ≤ 0.05 level of significance.

Morphological and morphometric analyses
Analysis of livers showed decreased Weight and size in BP group against control. Same findings were in case of kidneys, significantly reduced weight were recorded in BP treated group in comparison with control. Organ weight in vehicle was lowered but not significantly different from control. Turmeric has shown recovery in above mentioned parameters to a great extent. Morphometric calculations of livers and kidneys are given in Table 1.

Biochemical analysis of serum
Blood serum analysis of BP treated group has shown significant differences in comparison to control and antidote group. LFT and RFT are specified indicators for the hepatic and renal functions. Levels of ALT, AST, ALP and bilirubin were raised whereas total protein concentration was lowered in BP treated group. Moreover, urea and creatinine levels were also high in BP treated group as compared to control (Table 1). But in BP+AD group when BP was given along with turmeric, enzyme levels were reversed to some extent but not completely, which showed the limitation of turmeric.

Histopathological observations of liver
Histopathological analysis of liver in butyl paraben administrated group has shown hepatic portal vein filled with red blood cells and necrotic cells. Group treated with butyl paraben along with turmeric has shown some ameliorative effects as turmeric has reduced the number of red blood cells in central vein. Hepatic tissues obtained from vehicle control and antidote groups were similar with control. Photographs were taken at 100X and 400X (Figure 1).

Histopathological outcomes of kidney
Histology of kidney in control group exhibited regular epithelial cell lining, glomerulus, bowmen capsule and proximal convoluted tubules. Whereas renal hemorrhages were seen along with disruption of nuclei, unhealthy epithelium and broad bowman's space in BP administered group. Antidote ± dose group had shown reduced renal lesions and normal glomerular morphology. Animals of vehicle and antidote groups showed no damaging effects and were similar to control group. Photographs were taken at 100X and 400X (Figure 2). Contemporary age of industry is rich with many chemicals and compounds that are harmful to human beings. Their ceaseless use in certain commercial and personal products enhances their concentration in both biotic and abiotic environment (Dellafiora et al., 2017). Butyl paraben is an environmental toxicant used in food products, cosmetics and pharmaceutical industry mainly as preservative.
In present study, weight of liver and kidney reduced when exposed with butyl paraben at sub chronic level. There was difference in organs weights as compared to control by using only corn oil. Our results are in accordance with (Nowak et al., 2018) who reported peanut oil and corn oil as potent factors for weight loss. Oral administration of butyl paraben indicated effects on liver and kidney because it disturbed the absorption of food and metabolism, resulted a significant decrease in liver and kidney weights. Exposure to butyl paraben reduced the essential currency of cell i.e. ATP production by mitochondria. This lead to weight loss of organs as also reported by Boberg et al. (2016); Garcia et al. (2017) studies. Morphometric analysis has also shown that length and width of liver and kidney was lowered in BP group as compared to others and use of turmeric has improved these effects to much extent.
Increased concentrations of hepatic enzymes due to action against the harmful effect of butyl paraben were recorded in current study and these results are also reported by Darbre et al. (2008). Total protein concentration was reduced whereas bilirubin level in plasma was increased.
In case of kidney, urea and creatinine concentrations in serum of BP group were high as compared to (BP+AD) and control groups and it showed improper glomerular filtration in kidneys. These findings were corroborating with observations of Wahlang et al. (2013). As bilirubin levels were high due to the malfunctioning of hepatocytes to remove the metabolites of heme, this also effected the working ability of kidney cells and glomerular filtration rate, so resulted in high levels of urea and creatinine. In normal conditions creatinine is produced as a metabolite of creatine and is processed by kidney and excreted out by urine but when kidneys are not working properly due to any exogenous and endogenous factors, its concentration gets increased (Harvey et al., 2006). Elevated levels of hepatic enzymes, ALT, AST, ALP and Bilirubin in BP exposed group, as reported in our study was an indication of liver malfunction caused by paraben. This is similar to a lot of studies that state, due to any exogenous or endogenous factor like toxic compounds or viruses, hepatic enzymes balance is disturbed (Abbas et al., 2010). These enzymes are accountable for many metabolic reactions in the hepatic cells. They assist in detoxification of poisonous compounds and also confer antioxidant mechanism. 1: shows the effect of Butyl paraben on various morphometric and biochemical parameters of liver and kidney in female mice.