Hepatoprotective Effect of Costus Afer on Trace Metal Mixture Treated Rats Mediated by Regulation of Oxidative Stress Markers, Inammatory Cytokines and Bio-Metal Chelation

Technological developments have led to exposure to various substances that are harmful to the environment and public health, including heavy metals. In the environment, these grades of metals are usually diverse mixtures shown to cause physiological, biochemical and neurological dysfunctions in humans and laboratory animals. Cadmium, Lead, and mercury have been envisaged to exhibit their hepatotoxic effects by oxidative induction damage and synthesis of reactive oxygen species (ROS). The current work evaluated the protective activity of aqueous leaf extract of Costus afer (ALECA)) on liver damage arsing from exposure to trace metal mixture (TMM): cadmium chloride (CdCl 2 ), lead chloride (PbCl 2 ), and mercury chloride (HgCl 2 ). Five groups of weight matched Sprague Dawley rats were treated for 90 days. Metal mixture and deionized water were used to treat the 2 groups of rats whereas the other 3 groups were treated with various doses of the ALECA through oral gavage alongside the metal mixture. Hepatic function parameters, oxidative biomarkers, inammatory cytokines, Morphological changes and trace metals (concentrations were monitored in the liver). TMM treatment resulted in signicant increase in ALT, AST, ALP, bilirubin, IL-6, MDA, but decreased albumin, total protein, IL-10, SOD, CAT and GSH levels. TMM also caused some morphological changes and increased the heavy metal (Pb, Cd and Hg) concentrations in the liver. The leaf extract gave a reasonable protective effect on the hepatotoxicity caused by trace metal mixture - through the mechanisms of metal chelation, anti-inammatory, and antioxidant although this depends on the dosage to the rats. ALECA may be benecial in the management of liver toxicity.


Introduction
Trace metals occur from both natural and human activities. Their continued use in the industry, agriculture, medicine and technology has led to a great concern of safety for human health and the environment (Tchounwou et al. 2012). Metals are systemic toxic agents known to cause several organs damage seven at low exposure levels. Lead, Mercury, and Cadmium affect human organs leading to hepatotoxicity (Rana et al. 2018). These elements are ranked among the precedence metals that are of enormous concern to the public health (WHO, 2010).
Uptake and elimination of extraneous compounds are the vital functions of the liver (Anyanwu et al. 2018). Hepatotoxicity refers to impairment to the hepatocyte ensuing from overload of chemicals and exogenous compounds which include heavy metals and their metabolites (Singh et al. 2011). Owing to the dominance of these metals in our ecosystem, the long-term effect of a combination of lead, cadmium and arsenic have been studied and liver toxicity recorded by Bhattacharjee et al (2016).. Treatment of rats with Pb and Cd combination have also resulted in liver damage (Yuan et al. 2014).
Our perceptive of the unfavorable health effects of metals is based fundamentally on studies conducted on persons with rather high exposure, such as in the metal industry or in densely polluted environments. Humans are pretty exposed in their homes and workplace. There is a need to carry out further studies on the consequences of chronic environmental trace metal and their mixtures animal models that can be extrapolated to humans (Kentson et al. 2018). Due to the necessity of mimicking real-life situation, it becomes important to evaluate the toxic consequences of various trace metal exposures.
The chelators from modern medicine have been effective in treating the acute toxicity of metal poisoning, elimination of metal from soft tissues, and formation of non-toxic complexes. Despite their contribution in the treatment of metal poisoning, they also have some drawbacks which include toxicity issues, availability and affordability concerns. These disadvantages constitute the need to provide effective and safe pre-treatment therapy. The knowledge of the relevance of natural antidotes as chemo-preventive agents in metal poisoning being deemed as 'generally regarded as safe' GRAS, affordability and availability has gained traction recently.
Different pharmacological properties of Costus afer has given rise to the study of its safe bioactive compounds that may show promise as drug. Protection of the liver from noxious trace metal mixtures is considered worthwhile given the importance.. The anatomical proximity of the liver to the intestines predisposes the liver to toxic assault. Hepatotoxicity is usually characterized by increased membrane permeability and changes in enzyme levels. In a hydrocarbon-mediated liver damge, there was a substantial decrease in serum ALP and AST within normal at 400 mg/kg of aqueous leaf extract of Costus afer (ALECA) compared to rat treated with 200 mg/kg of the extract through oral gavage (Ukpabi et  .This study focuses on investigatingthe hepatoprotective action of aqueous leaf extract of Costus afer (ALECA) on male albino rats exposed to trace metal mixture (TMM) of lead, cadmium and mercury.

Harvesting of Costus afer
Samples of Costus afer were collected from a farmland in the University of Port Harcourt, Rivers State, Nigeria, in an area free from air pollution due to vehicular tra c. Mr. A. O. Ozioko, who is a staff of Botany Department, University of Nigeria, Nsukka helped to verify the plant for its authenticity prior to its usage.
Preparation of the Costus afer leaf extract Leaves of Costus afer were washed to remove sand particles, pulverized and stored. 250g of the pulverized leaf samples were macerated in 500ml deionized water i for 24 hours amidst continuous agitations after the method of . The mixture was shaken while the pulverized leaves pressed to obtain the extract which was stored in a refrigerator at 4 0 C (Anyanwu et al. 2020a). The process was repeated after every four days of treatment to obtain fresh extract to replace the redundant extract over the 90 days treatment period (Anyanwu et al. 2020a).
Determination of the aqueous leaf extract of Costus afer (ALECA) dosage A total of 12 rats separated into four equivalent groups that received Costus afer 1000 , 2000 , 4000 5000 mg/kg respectively and were observed for 24 hours for any change in physical features or death. At the end of the treatment, no death or change in physical features was recorded. This implied that Costus afer has no toxic effect.
To determine the dose used for this study, an assumed dose was used since the extract was con rmed to be relatively safe. Thus, we assumed the dose of 3000 mg/kg and then used 25% of the assumed dose as the low dose, 50% of the assumed dose as the medium dose and 75% of the assumed dose as the high dose.

Animal care handling
The study used thirty-ve male albino rats that were about 8 weeks old with 100-200g weights procured from the Animal House of the Faculty of Pharmacy, University of Port Harcourt, Nigeria. The study utilized the animal husbandry procedure established in previous studies by  and Anyanwu et al. (2020b).

Design of the experiment
Five groups of seven rats each based on their weight were used for this study. The rst group was as the standard control on which only deionized water was administered whereas the second group received

Necropsy
After 90 days, the rats were anaesthetized with and sacri ced. The samples were collected according to the procedures recorded by Anyanwu et al. (2020a;2020b).

Preparation of liver homogenate
Liver tissue was homogenized inbuffer (pH 7.4). The supernatant was collected after centrifugation (3000 rpm for 15mins at 4 0 C) for in ammatory and antioxidant analysis.

Metal analysis
About 6ml and 2ml of Nitric acid and perchloric acid respectively were used for the acid digestion of the liver after isolating the weighed organ. The samples were left for 30 mins after acidi cation before being heated at 105 0 C until digestion was completed. Whatmann lter paper Number (1) was used for ltration so as to obtain clearer samples. The solution was later made up to 15 ml ( nal volume) with distilled water. All the glassware were thoroughly washed and rinsed before use. Calibration curves of Pb, Cd and Hg as previously described by Anyanwu et al. (2020a;2020b). Solar thermo elemental ame Atomic Absorption Spectrometer (Model SG 71906) was used to determine the levels of Pb, Cd and Hg at a detection limit of <0.001 mg/kg. Standard operating parameters were set and the hollow cathode lamps for Pb, Cd and Hg (Model SG 71906) were employed as radiation source and fuel was air acetylene. All the samples and standard were run in duplicate.

Hepatic biomarkers
Estimation of alanine aminotransferase (ALT) and Aspartate transaminase AST The alanine aminotransferase (ALT) and Aspartate transaminase AST activities of the liver samples were assayed employing a Randox kit (Reitman and Frankel 1975).

Estimation of ALP
The ALP function was determined with the aid of standard diagnostic kits (Randox Laboratories Ltd, UK) using the calorimetric endpoint (Klein et al., 1960).

Estimation of total and direct bilirubin
This was done using standard diagnostic kits in a calorimetric process (Randox Laboratories Ltd, UK; Jendrassic and Groff, 1938)

Estimation of total protein and albumin
This was done using a standard diagnostic kits Antioxidant analysis CAT activity CAT activity was assayed by adapting the method of Clairborne (1995).

Estimation of hepatic glutathione (GSH) level
Glutathione (GSH) level was estimated after the method of Sedlak and Lindsay (1968) .

Estimation of SOD activity
Following the method of Misra and Fridovich (1972), superoxide dismutase was determined.
Lipid peroxidation marker (MDA) activity Following the method of Ohkawa and Ohishi (1979), the MDA was evaluated.

Results
The phytoconstituents of aqueous leaf extract of Costus afer (ALECA) is shown on Table 1. had decreased liver weight ( Table 2). The absolute and relative weightsof liver of rats that received only trace metal mixture (TMM) was 9.4 ± 1.35 g and 3.62 ± 0.47 % respectively while the rats administered with only deionized water had 5.08 ± 0.95 g and 2.45 ± 0.41 % respectively. The tests for liver function were done to evaluate the likely protective role of Costus afer treatment from metal mixture exposure. Treatment with trace metal mixture (TMM)caused signi cant elevation in aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels, bilirubin (total and direct), and a decrease in total protein and albumin, whereas the rats that received both aqueous leaf extract of Costus afer (ALECA) and trace metal mixture (TMM) showed reduction in the liver enzyme markers and a rise in total protein and albumin in comparison to metal mixture-treated albino rats ( Fig. 1). The aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, total bilirubin, direct bilirubin, total protein and albumin levels in rats treated with only trace metal mixture (TMM) were signi cantly different (172 µ/l, 77.8µ/l, 222.2µ/l, 32.6 mg/dl, 15.2 mg/dl, 43.8 g/l, 25.4 g/l, p < 0.05) respectively, from the groups that received both aqueous leaf extract of Costus afer (ALECA) and trace metal mixture (TMM).  Table 3 whereas the correlation plot is shown in Fig. 3. An illustration of the differentiation of parameters and the interactions among liver function parameters in different groups is depicted in Fig. 4. A 3-component system showing 95.35% of total variance was obtained after statistical principal component analysis.
The co-ordinate plot of liver function parameters showing the association between the variables consists of 3 classes. The result showed that class 1 rats had high total protein and albumin levels with low aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, total bilirubin and direct bilirubin,, while class 2 rats had high aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, total bilirubin and direct bilirubin levels with low total protein and albumin levels. Strong positive correlations (≥ 0.88) were perceived between these parameters (aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, total bilirubin and direct bilirubin) whereas a weak association (r = 0.6617) was observed between the Total protein and albumin. Also, both factors had negative correlations with aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, total bilirubin and direct bilirubin which is an indication that a reduction in total protein and albumin would likely lead to an increase in aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, total bilirubin and direct bilirubin and vice versa. All the variables were printed in bold in PC1 (Table 2) whereas aspartate aminotransferase had the highest loading on PCI after Varimax rotation. The principal components were extracted with the aid of a 3D graphing software to plot the correlated variables. An assessment of the in ammatory status after treatment with trace metal mixture TMM was done by evaluating the pro-and anti-in ammatory cytokines level in the liver. The co-treatment using Costus afer signi cantly decreased the levels of pro-and also increased (p < 0.05) the anti-in ammatory cytokines (IL-6 and IL-10) in the liver tissue in comparison to in the trace metal mixture TMM-treated group (Fig. 5), suggestive of anti-in ammatory activity of aqueous leaf extract of Costus afer (ALECA). The pro-and anti-in ammatory cytokine levels (IL-6 and IL-10 respectively) of rats administered with the trace metal mixture TMM only was signi cantly different (61.8 Pg/g tissue and 14.7 Pg/g tissue, p < 0.05) from the in ammatory cytokines seen in rats co-treated with Costus afer.
The oxidative status in the hepatocyte subsequent to the metal mixture treatment using the lipid peroxidation marker, MDA level was evaluated. The 90-day treatment which was done with the following metal mixture in the following dosage PbCl 2 -20mg/kg, CdCl 2 -1.61mg/kg, HgCl 2 -0.40mg/kg body weight induced oxidative reaction in the organ (liver tissue). The MDA level increased signi cantly (p < 0.05) in the hepatocyte of trace metal mixture TMM -treated rats in comparison to those of the normal control group (Fig. 6). A signi cant decrease in oxidant level was observed in the rats treated with Costus afer and metal mixture compared to those treated with only the metal mixture.
With respect to how the treatment affects the non-enzymatic glutathione (GSH) and enzymatic superoxide dismutase and catalase (SOD and CAT) activities in the liver tissue. Treatment with trace metal mixture TMM resulted a signi cant decrease (p < 0.05) in glutathione and superoxide dismutase and catalase levels in comparison with control. Rats that received aqueous leaf extract of Costus afer (ALECA) plus trace metal mixture TMM had elevated levels of glutathione and superoxide dismutase and catalase in comparison to rats that received only trace metal mixture TMM. s Heavy metal concentration on the liver of the rat samples The concentration of trace metals (lead, cadmium and mercury) in the liver were notably elevated (p < 0.05) in the liver of the rats treated with the trace metal mixture TMM-in comparison to the control ( Table 4). Treatment of rats with aqueous leaf extract of Costus afer (ALECA) plus trace metal mixture TMM resulted in signi cant reduction in trace metals (lead, cadmium and mercury) levels in comparison to rats that received only trace metal mixture TMM In addition, the group exposed to the trace metal mixture TMM only, had the highest level of trace metals (lead = 90.992 ± 13.284, cadmium = 0.78 ± 0.133 and mercury = 0.305 ± 0.0439) in comparison to the control group. Pearson's rank correlation analyses indicate the inter-trace metal relationship among trace metals in liver of rats showed strong positive correlation (r > 0.90) between metals such as: Pb and Cd, Pb and Hg, as well as Cd and Hg. All correlations were signi cant at p < 0.01 (Fig. 7).

Regression Model Development
XLSTAT 2016 software was employed for the model development in this study. Lead, Cadmium, Mercury, and ALECA were considered as the input data and were used for the calibration of the model using multiple linear regressions. Zero coe cients for x 2 and x 3 were observed as outputs and thus subsequent calibrations were done without the 3 constant parameters (input data).. In other words, y is made a function of ALECA variable for a given set of the heavy metal variables namely: Lead, Cadmium, and Mercury. A plot of catalase concentration vs ALECA concentration was modelled using linear, quadratic and exponential options ( Table 5). The R 2 , MSE and RMSE values were determined and shown in Table 6 for the process.
For veri cation of he calibrated models, the observed model was compared with predicted model on the liver catalase level ( Fig. 9) with corresponding R 2 = 0.935 .
The quadratic and exponential models for the liver catalase level was R 2 , MSE and RMSE of (0 .180 respectively. Similarly, the veri cation was equally done on other parameters in the liver.. These models could help predict the residual values of liver function parameters, antioxidant pro le and in ammatory cytokines in the liver of male albino rats at any treatment dose using ALECA to a high precision provided the levels of lead, cadmium, and mercury remains constant as considered in this work. A replication of regression models was done on other biomarkers and the best with respect to R 2 , MSE and RMSE values was selected and summarized as shown in the Table 6 below;  Many previous studies described trace metals as immune-suppressors that result in elevated levels of TNF-α, Il-1β and IL-6(pro-in ammatory cytokines). The elevated level of pro-in ammatory cytokine  in this study may be due to heightened production of reactive oxygen species (Gao 2012;Guyot et al. 2015). Oxidative stress could be associated with an excessive production of the pro-in ammatory cytokines (Gao 2012  Superoxide dismutase is the rst defense interval for the conversion of superoxide radical anion to the production of free radicals involved in hydrogen peroxide, whereas catalase serve as the second antioxidant protection mechanism by reducing hydrogen peroxide to oxygen and water (Younus, 2018).
Thus, aqueous leaf extract of Costus afer (ALECA) increased antioxidant enzymes levels which is in agreement with the previous study protective effects of Costus afer on the liver by .
Despite the fact that the exact mechanism of the metal mixture toxicity is unclear, the observations in this study indicates that treatment with trace metal mixture TMM elicits reactive oxygen species (ROS) generation and impairment cellular antioxidant capacity. This could result in an imbalance between free radical species and the body's resistance against cellular damage (Koivula and Eeva 2010). Therefore, supplementation of antioxidant molecules would be exogenously bene cial in cell antioxidant protection to neutralize heavy metal poisoning. Aqueous leaf extract of Costus afer (ALECA) contains antioxidant phytochemicals such as phenolics and avanoids (Ezejiofor et al.2017). This study has shown the protective effect of aqueous leaf extract of Costus afer (ALECA) over trace metal mixture TMM -induced toxicity in rats.
Malondialdehyde, a marker of lipid peroxidation, showed a substantial increase (p < 0.05) in the liver of rats that received trace metal mixture TMMin comparison with groups that received aqueous leaf extract of Costus afer (ALECA) plus trace metal mixture TMM. Malondialdehyde is a marker used to measure the level of oxidative stress in an organism (Singh et al. 2014 in ammation and distortion of histoarchitecture of the liver in rat model. The observed attenuation of destructive effects of TMM by effects tended to be attenuated by the aqueous leaf extract of Costus afer (ALECA) which could proffer some hope as an alternative remedy and circumvent the major drawbacks of notable chelators: increase in toxicity, inaccessibility and high-priced associated with modern medicine. Therefore, Costus afer could be a potential hepatoprotective agent.
Declarations Figure 1 Effect of Costus afer extract on serum hepatic enzymes of male albino rats treated with metal mixture.  Inter-trace metal correlation among trace metals in the liver of rats showed strong positive correlation (r > 0.90) between metals such as (a) Cd and Pb (b) Hg and Pb (c) Hg and Cd during the study. All correlations were signi cant at p< 0.01.  Predicted liver catalase level against observed liver catalase level (nMole/mg tissue); where y = Catalase levels; x = ALECA concentrations served as input values.

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