Involvement of Bitter Leaf Extracts in Testicular Metabolic Stress Induced by Petroleum Tainted Diets in Rats

Introduction

The environment in which we live has increasingly been inundated with several types of environmental toxicants which has the tendencies of causing injuries and metabolic stress to plants, animals and humans alike. Several industrial pollutants which includes unrefined petroleum and its allied products such as kerosene, flared gases, premium motor spirit, diesel, 3,1-dinitrobenzene or nonylphenol, methanoxyethanol, glycol ether and brake oils are known to exert testicular oxidative metabolic stress and atrophy. One significant danger of those who are exposed to environmental toxicants is the increased risk of being infertile which has been defined as the inability of a person or couple who is sexually active and a non user of contraceptive to achieve spontaneous pregnancy within one year (WHO, 2010; Zegers et al., 2009). Ample evidences from studies reveal that most male infertility problems are a result of testicular oxidative stress which has been reported to affect seminal plasma antioxidants increased lipid peroxidation (alteration of sperm morphology, impaired sperm motility.

The mechanistic defense against oxidative stress depends on the ability of the body and cells to boost the buffering capacities of antioxidants which will help in clearing of oxidative radicals generated from various metabolic processes especially when it relates with the clearance of toxicants. Today, Vernonia amygdalina which is a well known vegetable common to many tribes of Nigeria has been elucidated for its antioxidant buffering capacities. Vernonia amygdalina is well known for its use as an alternative regimen for malaria. It has been used severally as a protective and ameliorative agent for the deleterious effects of many toxicants such as cyanide, carbon tetrachloride, unrefined petroleum and cycasin.

From the foregoing, there is no doubt that there exist ample evidences on the ability of unrefined petroleum to induce various forms of metabolic oxidative stress, there exist little evidence on the possible role of unrefined oil intoxication to induce testicular damage occasioned by unrefined petroleum adulterated feeds as well as the ability of Vernonia amygdalina to induce the possible restoration or control of activated metabolic stress parameters. This study therefore was carried out to clearly show research evidences to cover these existing gaps.

Materials and Methods

Matured bitter leaf (Vernoniaamygdalina Del) was collected from a farm at Abraka, Nigeria and preliminary identification carried out at the Department of Botany, Delta state University, Abraka, Nigeria by Dr Erhenhi A. H. The leaf was later authenticated at the Forestry Research Institute of Nigeria, Jericho Hill, Ibadan, Nigeria, were a specimen with the voucher number, F101963 was deposited at the herbarium. Male albino rats (Rattusnorvegicus), thirty six, an average weight of 150g-182g were acquired from the animal house at the Delta State University, Abraka Nigeria. The rats were accommodated in a wooden cage and allowed to acclimatize for one week on grower’s mash (a product of Rainbow Feed Limited). The composition of the feed as declared by the manufacturer was previously published by Achuba (2018). All other reagents used for biochemical assay were of analytical grades.

Bitter Leaf Extract Preparation

The bitter leaf was washed, chopped and air dried at room temperature for one week in an open space within the laboratory of the Department of Biochemistry, Delta State University, Abraka. After drying, the bitter leaf was chopped off and macerated using a warren blender to a smooth dry powder. The bitter leaf extract was prepared using methanol as described by Yin et al. (2013). One hundred (100g) of the powdered leaf was dissolved in 400ml of methanol through sonication for 10mins, then filtered with Whatman No.1 using vacuum pump. The extract obtained concentrated via rotary evaporator at 40-50℃ under reduced pressure to get the bitter leaf methanol extract (BLME). The extract was stored at - 8℃ until required.

Experimental Design and Treatment

The distribution of six rats per group was done according to the following description:

Group A = Feed

Group B = Feed +100 mgkg-1body weight of BLME

Group C = Feed+200 mgkg-1body weight of BLME

Group D = Feed (100g Feed+4ml unrefined Oil)

Group E =Feed (100g Feed+4ml unrefined Oil) +100 mgkg-1body weight of BLME

Group F= Feed (100g Feed+4ml unrefined Oil) +200 mgkg-1body weight of BLME

The bitter leaf extract used was freshly prepared at the point of administration. To obtain to obtain 200 mgml-1 20 gram of the extract was dissolved in 100 ml of distilled water out of which aliquots of the freshly dissolved extract was administered by gavage according to the rats body weight once daily. The rats in groups A and D were not administered the extracts while all rats were allowed free access to water. All the treatments lasted for a period of 30 days

Sample Collection

After 30 days exposure period, the rats were sacrificed via cervical decapitation on the 31st day after an overnight fasting. The testes were collected into pre-chilled labelled containers. Testes wet tissue (0.5g) was homogenized with 9.0 mL of normal saline using pre-chilled mortar and pestle and the supernatant obtained was stored at -8C in the cold room and used for biochemical analysis within 48 hours.

Biochemical Analysis

Standard methods were employed for the assay of level of lipid peroxidation (MDA) (Gutteridge and Wilkins, 1982) and enzymatic oxidative stress markers as follows; aldehyde oxidase (AO) (Omarov et al. 1998), sulphite oxidase (Macleod et al. 1961); monoamine oxidase (MO) and xanthine oxidase (XO) (McEwen, 1971). Assay for the non-enzymatic antioxidant profile in the testes were determined using the methods of Ellman (1959) for assay of reduced gluthathione while Assay for vitamin C employed the methods reported by Achuba (2008). Assay for specific activities of enzymatic antioxidants were carried out employing the methods of Misra and Fredorich (1972) for superoxide dismutase (SOD), Cohen et al., (1972) for Catalase, Habig et al. (1974) for gluthatione-s-transferases (GSTs) and Khan et al. (2009) for glutathione peroxidase (GPx).

Histological Examination

A known portion of the testes, of each rat were harvested and fixed in 10% formol saline for 48hours and processed for paraffin wax embedding with an automatic tissue processor by dehydrating through 70%, 90%, 95% and two changes of absolute ethanol for 90 minutes each. Clearing was achieved through two changes of xylene for 2 hours each; and infiltrating with two changes of paraffin wax for 2 hours. Sections were cut at 5μm with a rotary microtome. The sections were stained by haematoxylin and eosin (H and E) using the method of Odoulaet al. (2009), examined and photographed using a light microscope.

Statistical Analysis

Analysis of data was carried out using the single factor analysis of variance (ANOVA) with the aid of the Statistical Package for the Social Sciences version 17 (SPSS 17). Post hoc analysis (comparisons across Groups) was done using Bonferroni at P <0.05 level of significance.

Result

Result presented revealed a significant rise in lipid peroxidation (MDA) levels in rats administered both doses of BLME without tainted diets (B and C) relative to positive control (A) which was fed normal diets. This did not differ significantly with rat fed tainted diets without treatment ( groups D) and rats fed tainted diets and administered both doses of the BLME( groups E and F). Feeding rats with unrefined oil tainted diets. Also, there were observed increase in the activities of AO, SO, MO and XO of rats administered both doses of BLME in groups B and C relative to rat fed with untreated feed (group A) but significant reduction relative to rats fed with unrefined petroleum tainted feed (group D). Administration of both doses of BLME to rats fed with unrefined petroleum tainted feed (groups E and F) significantly increased the activities of the oxidases (AO, SO, MO and XO) relative to the rat fed with untreated feed (group A) and rats fed with unrefined petroleum tainted feed (group D). However there were no significant difference in groups E and F when relative together

As shown, the activities of CuZnSOD did not significantly increase in rats administered 100mgKg-1 body weight of BLME (group B) relative to normal control group A fed with only normal diets. It however significantly increased in rats administered 200mgKg-1 body weight relative to control. Also, the CuZnSOD activities in rats administered both doses were significantly higher than in the rats administered petroleum tainted diets without treatment and those fed tainted diets and treated with both doses of BLME. Rats fed with only tainted diets were observed to have reduced CuZnSOD activities relative to normal control but significantly increased relative to those fed tainted diets and treated with 200mgKg-1 body weight. The activities of MnSOD did not change in rats in groups A and B but increased significantly when rats in group C and group A are relative. The activities of MnSOD significantly reduced in rats fed with tainted diets in group D relative to normal control and rats fed normal diets and treated with both doses of BLME in groups B and C. Treatment of rats fed tainted diets with 100mgKg-1 and 200mgKg-1 of BLME showed no significant difference relative to the untreated rats in group D. Total SOD activities showed no significant difference across groups A-E but significantly reduced in group F which was exposed to tainted diets and treated with 200mgKg-1 of BLME relative to groups A-D.

Results presented reveal that there were no significant change in levels of Vitamin C across all groups. GSH levels were observed to have no significant change in rats administered 100mgKg-1 and 200mgKg-1 body weight of BLME (B and C) relative to normal control group A but significantly increased relative to rats fed petroleum polluted diets. Administration of 100MgKg-1 body weight of BLME increased significantly GSH levels relative to those fed only tainted diets but reduced relative to normal control (group A). Those fed polluted diets and administered 200mgKg-1 body weight of BLME (group E) remained unchanged relative to group D but reduced relative to all other groups. The activity of the antioxidant enzyme catalase was significantly elevated in rats treated with 100mgKg-1 body weight but not with 200mgKg-1 relative to control group A. This was however increased significantly for both doses relative to rats in group D fed tainted diets without treatment. treatment with both doses led to a further reduction in catalase activities relative to groups A and D. GPX and GSTs activities were observed to have no significant change for rats treated with 100mgKg-1 body weight without contamination relative to control (group A) and reduced significantly for GPX while increasing for GSTs relative to rats fed only polluted diets (group D). Treatment with both doses of BLME significantly reduced in GSTS activities relative to the control group A while GPx activities were only significantly reduced for the 200mgKg-1 body weight dose. Relative to group D however, it was observed that GSTs activities remained significantly unchanged both doses (100mgKg-1 and 200mgKg-1) body weight. GPx on the other hand reduced significantly for both doses.

Discussion

Contamination of unrefined petroleum has remained a significant contributor to several endocrine induced metabolic stress and malfunction. Testicular oxidative stress on the other hand is said to be responsible for a majority of the many cases of infertility world over. The result presented in this study revealed increased levels of MDA and the activities of the oxidative enzymes (AO, SO, MO and XO) in the testes of rats fed tainted diets relative to normal control. Rise in MDA levels have been increasingly reported as a potent marker for the negative effects of consuming unrefined petroleum diets and other unrefined oil allied exposures. Petroleum induced rise in peroxidation of tissues are said to go concurrently with eventual rise in oxidative enzymes which are needed to initiate eventual clearance of the peroxides and super oxides generated by petroleum contamination.

It is important to state that based on the physiological position and nature of the testes, it is said to be highly vulnerable to toxins hence there exist an inbuilt enhanced antioxidant buffering due to the presence of non enzymatic antioxidants (vitamins such as vitamin C, vitamin E and GSH) and the enzymatic antioxidants (SOD, GST, GPX and catalase). Therefore, for oxidative damage to occur, there must be evidence based overwhelming of the antioxidant defence capacities of the tissues and biological membranes involved. The observed induction of lipid peroxidation and the oxidative enzymes in the testes of rats fed petroleum polluted diets without treatment with BLME are in concordance with the observed reduced levels of the antioxidant defensive markers SOD and Catalase, GPx, GSTs, GSH and Vitamin C. These observations are similar to earlier observations made by Achuba et al., (2016); Achuba (2018a) and Ita et al. (2018). The administration of BLME to these rats was not able to reverse to a comparable status the levels of these non-enzymatic and enzymatic antioxidants relative to the control which was not fed with petroleum tainted diets. These observations are not in line with earlier submissions made by Ita et al, (2018) , Achuba, (2018) and Okpoghono et al., (2018) who reported the abilities of Ageratum conyzoides, Vernonia amygdalina and Monodura myristica to successfully mitigate the rising metabolic stress in the testis, kidney and liver of rats fed petroleum tainted diets respectively. This observed trend thus indicates the inability of the BLME to control the oxidative and metabolic balance of the testes in the presence of petroleum hydrocarbons.

Also, this study observed a concomitant rise in testicular lipid peroxidation, the antioxidant defence, and the activities of the oxidative enzymes in rats administered 100 mgKg-1 body weight and 200 mgKg-1 body weight of the BLME without exposure to unrefined oil polluted diets. This observation also further substantiates the earlier claim of the possible noxious effects of Vernonia amygdalina on the testes which has been previously implicated to have a possible anti spermatogenic effect and reduction in testosterone levels, sperm motility and sperm count, seminiferous tubular diameter, cross sectional area, numerical densities of seminiferous tubules, number of profiles per unit area and increased toxicological profile of the testes. The possible justification of this observation may be likened to adverse drug reactions and interactions that occur during drug and xenobiotic metabolism (Barnerjee et al., 2016; Gandhi et al., 2012). This assertion is claimed because there are ample research evidences that submits that in the course of drug and xenobiotic biotransformation, that certain enzymes such as the lipoxygenases, cyclooxygenase and the oxidases have the capacities of developing oxidative radicals which in turn contributes to the pool of ROS present in the tissues that eventually shuts down their antioxidant defence systems by the depletion of these enzymes and other antioxidant enzymes.

There is no doubt that findings in this study indicate testicular hypertrophy and autophagy in the rats. The observed distortion in testicular architecture are similar to those reported by Salau et al., (2013) and Oyedeji et al., (2013) thus substantiating further the possible contribution of Vernonia amygdalina to all the observed metabolic stress reported in this study.

Conclusion

This study has shown that the consumption of petroleum tainted diets contributed to the induction of testicular metabolic stress in experimental rats. However, treatment with Vernonia amygdalina methanolic extracts could not reverse the observed metabolic stress but contributed to the increasing levels of metabolic stress in the testes and the alteration of the testicular ultrastructure. This thus gives an insight into the possible toxic effect of Vernonia amygdalina on the ability to induce infertility in males. Based on this therefore, it is submitted that there is need for further research to understand the possible mechanism and molecular bases for the observed alteration of theses metabolic stress in the testis owing to the proven records of the antioxidant buffering capacity of Vernonia amygdalina in other tissues.