INVESTIGATION INTO THE EFFECT OF THE TOXICANTS ARSENIC AND MANGANESE ON MALE REPRODUCTIVE SYSTEM OF WISTAR RAT.

TABLE OF CONTENTS

CHAPTER ONE

1.1       INTRODUCTION.

1.2       LITERATURE REVIEW

1.2.1     ARSENIC

1.2.2    PHYSICAL AND CHEMICAL PROPERTIES OF ARSENIC.

1.2.3    MANGANESE

1.2.4    PHYSICAL AND CHEMICAL PROPERTIES OF MANGANESE

1.2.5    TOXICITY OF ARSENIC AND MANGANESE

1.2.6      MECHANISM OF ACTION OF ARSENIC

1.2.7    ABSORPTION AND METABOLISM OF ARSENIC

1.2.8    TOXICITY OF MANGANESE

1.3.0    EPIDERMOLOGY OF ARSENIC AND MANGANESE WORLDWIDE

1.3.1    EPIDIDYMIS

1.4.0    FREE RADICAL AND OXIDATIVE DAMAGE

1.4.1    BASIC TYPES OF DAMAGES CAUSED BY FREE RADICALS

1.4.2    FORMATION OF FREE RADICALS

1.4.3          REACTIVE OXYGEN SPECIES

1.4.4    LIPID PEROXIDATION.

1.4.5    OXIDATIVE STRESS

1.4.6    ANTIOXIDANTS

1.5       AIM AND OBJECTIVES

CHAPTER TWO

MATERIALS AND METHODS

2.1       CHEMICALS

2.1.2    EXPERIMENTAL ANIMALS

2.1.3    ANIMAL TREATMENT

2.1.4    EXPERIMENTAL DESIGN

2.1.5    SACRIFICING OF EXPERIMENTAL ANIMALS AND COLLECTION OF EPIDIDYMIS

2.1.6    COLLECTION OF BLOOD

2.1.7    HOMOGENIZATION OF THE EPIDIDYMIS

2.1.8    REAGENTS PREPARATION

2.2       BIOCHEMICAL ASSAYS

2.2.0    DETERMINATION OF PROTEIN CONCENTRATION

2.2.1    INDUCTION OF OXIDATIVE STRESS (LPO ASSESSMENT)

2.2.2    DETERMINATION OF CATALASE ACTIVITY

2.2.3    DETERMINATION OF SUPEROXIDE DISMUTASE (SOD) ACTIVITY.

2.2.4    ESTIMATION OF REDUCED GLUTATHIONE (GSH) LEVEL

2.2.5                ESTIMATION OF GLUTATHIONE-S-TRANSFERASE (GST) ACTIVITY

2.2.6    DETERMINATION OF HYDROGEN PEROXIDE CONCENTRATION

2.7       STATISTCAL ANALYSIS

CHAPTER 3

RESULTS

1.1              Average water intake of animals exposed to Arsenic and Manganese.

3.2         Average body weight gain of animals exposed to Arsenic and or Manganese

3.3       Epididymal weight gain of animals exposed to arsenic and or manganese.

3.4          Relative epididymal weight gain of animals exposed to arsenic and or manganese.

3.5        Initial and final body weight gain of animals exposed to arsenic and or manganese.

3.6    Initial and final weight of animals at the recovery phase of the study

3.7       Reduced glutathione level in epididymis of animalsexposed to arsenic and manganese

3.8      Lipid peroxidation (LPO) in the epididymis of animals co-exposed to arsenic and

            Manganese

3.9     Hydrogen peroxide concentration in the epididymis of rats exposed to arsenic

                        and manganese

3.10    Activity of glutathione-S-transferase (GST) in the epididymis of animalsexposed

                                                                                                  to arsenic and manganese

3.11The activity of catalase in the epididymis of rats exposed to arsenic and or

manganese

3.12Activity of superoxide dismutase in the epididymis of rats exposed to arsenic 

and manganese

3.13   Sperm characteristics of animalsexposed to arsenic and manganese. 

   3.14                HISTOPATHOLOGY

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 DISCUSSION.

4.2   CONCLUSION

REFERENCES

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

                                                                                                                                                                                                                           1.1       INTRODUCTION.

In today’s industrialized world, exposure to pollutants in which heavy metals like arsenic, lead, manganese are an example is of high risk. These metals are present even in drinking water. Arsenic is mostly present in underground water. These metals are highly distributed in our environment and are thus consumed in quantities greater than what by the body requires (Ferrer, 2003).

Increased levels of arsenic in the environment, is an attribute to industrial product and waste, agricultural pesticides and herbicides. Although manganese is an essential element, toxicity can be gotten from drinking water, food, occupation and so on. Exposure to these heavy metals can cause poison and damage to models (the human body. Effects of arsenic have been reported in both human and experimental ATSDR a, 2012; Kannan et al., 2001). Mn exposure can also cause neurotoxicity (ATSDR b, 2007). Manganism, a consequence of exposure to high Mn levels, is a known neurological syndrome with many symptomatic analogies to Parkinson’s disease (Santamaria, 2008). Manganese and arsenic also target the same organ in the body, namely the brain (ATSDR, 2007a,b,c).

Given their co-existence in soil and atmosphere, exposure to toxicity does not occur in isolation (Kordaset al., 2010).   Indeed, in the real world, exposures to complex mixtures are the rule, rather   than exception (Scherer, 2005). Over the last several decades, the incidence of neurological diseases has increased (WHO, 2006).Mn poisoning results in an irreversible condition known as “manganism,’’ a neurodegenerative disorder that resembles Parkinson disease in both symptomatology and the underlying cellular mechanisms (Ellingsen et al., 2008; Martinez-Finley et al., 2012).

Neurological disorders induced by chronic metal exposure can be progressive and manifest clinically decades after the initial exposure (Gil and Pla, 2001). The onset of neurotoxic effects is largely subtle, insidiously manifested and unidentifiable as a clearly defined disease (Shy, 1993).

Exposure to arsenic- and lead-contaminated drinking water has been associated with an increased occurrence of congenital heart defects (CHDs). Groundwater is a vital hidden natural resource

(Tularam and Krishna 2009; Lashkaripour and Ghafoori 2011). Groundwater can be found in most environments and generally requires no prior treatment and can be found close to the points of demand often at low cost (MacDonald and Calow 2009). Arsenic poisoning or arsenicosis is a condition caused by the ingestion, absorption or inhalation of dangerous levels of arsenic, higher than the normal 10ppb which the body can tolerate.

The male reproductive system consists of two major parts: the testes, where sperm are produced, and the penis, according to Merck Manuals. The penis and urethra belong to both the urinary and reproductive systems in males. The testes are carried in an external pouch known as the scrotum, where they normally remain slightly cooler than body temperature to facilitate sperm production.Metals may cause a wide spectrum of reproductive and developmental adverse effects such as reduced fertility, abortions, retarded growth at the intrauterine cavity, skeletal deformities, malformations and retarded development especially of the nervous system.

 Arsenic and manganese tend to decrease motility of sperm in the male reproductive system even though the sperm are active.

The important mechanisms of action of arsenic are placental transfer, oxidative stress, direct binding with thiol group etc.

 The toxicity of arsenic in male and female reproductive organs is also explained. It also throws some light on the therapeutic strategies for metal toxicity.Manganese is a suspected reproductive toxicant and exposure to it has the potential to negatively affect the human reproductive system. The severity and nature of the adverse effect is variable and can be influenced by factors such as level of exposure and individual sensitivity to the chemical. Effects on the male reproductive system can include such things as altered sexual behavior, altered fertility and problems with sperm shape or count. 

Manganese also have some positive effects on the reproductive system, they include It helps to produce sex hormones and sperm. Manganese acts as a catalyst for breaking down fatty acids and cholesterol. Manganese has a positive effect on the male reproductive system,It also enhances the brain's aptitude for receiving and sending messages,Sex hormones are produced in the pituitary gland, where a considerable amount of manganese exists. Because of this, manganese is believed to assist in sexual health.

Studies have been carried out on the individual effect of manganese and arsenic on the male reproductive system, this research however concentrates on both their individual effect and also their combined effect on the reproductive system. Earlier studies have shown that both accumulate in the brain and affect production of hormones.

Apart from affecting the reproductive system of man, arsenic and manganese cause other side effect including cancer. Arsenic and manganese have been shown to induce oxidative damage in the membrane leading to production of free radicals that may induce cancer and apoptosis. On the other hand some studies have suggested that arsenic can aid cancer treatment as it assists blood thinning.
These studies however have not been confirmed.  The effects of arsenic and manganese can be assessed in male induced rats using assays like H202, Lipid Peroxidation, GSH, GST, SOD etc.

Pollution of the environment by these heavy metals is indeed a cause for alarm and have caused adverse effect to the human body as stated by WHO, unsuspected sources like underground water have shown lack of awareness by individuals.

1.2 LITERATURE REVIEW

Any foreign substance that enters the body is called xenobiotics. These substances can undergo any of the following pathways;

2.      Excretion from the body unchanged

3.      Undergo spontaneous reaction of its own

4.      Undergo metabolism.

Most xenobiotics undergo the third pathway, however if the body is over exposed to a compound it will induce its own reaction and might likely undergo the above second pathway. Arsenic and managanese are foreign compounds which enter the body through various means.

1.2.1        ARSENIC

Arsenic is a chemical element with symbol Asandatomic number 33. Arsenic occurs in many minerals, usually in conjunction with sulfur and metals, and also as a pure elemental crystal. Arsenic is a metalloid. It can exist in various allotropes, although only the gray form has important use in industry.A few species of bacteria are able to use arsenic compounds as respiratory metabolites. Trace quantities of arsenic are an essential dietary element in rats, hamsters, goats, chickens, and presumably many other species, including humans. However, arsenic poisoning occurs in multicellular life if quantities are larger than needed.

Arsenic contamination of groundwater is a problem that affects millions of people across the world (Mameli et al., 2001).

Arsenic and its compounds, especially the trioxide, are used in the production of pesticides, treated wood products, herbicides, and insecticides. However, these applications are declining. Arsenic can be found naturally on earth in small concentrations. It occurs in soil and minerals and it may enter air, water and land through wind-blown dust and water run-off (Martinez-Finley et al., 2012).

Despite its notoriety as a deadly poison, arsenic is an essential trace element for some animals, and maybe even for humans, although the necessary intake may be as low as 0.01 mg/day. Most arsenic is found in conjuction with sulfur in minerals such as arsenopyrite (AsFeS), realgar, orpiment and enargite. None is mined as such because it is produced as a by-product of refining the ores of other metals, such as copper and lead. A very high exposure to inorganic arsenic can cause infertility and miscarriages with women, and it can cause skin disturbances, declined resistance to infections, heart disruptions and brain damage with both men and women (Dhatrak and Nandi, 2009; Mejı´a et al., 1997).

Finally, inorganic arsenic can damage DNA. A lethal dose of arsenic oxide is generally regarded as 100mg. Organic arsenic can cause neither cancer, nor DNA damage. But exposure to high doses may cause certain effects to human health, such as nerve injury and stomachaches.

1.2.2           PHYSICAL AND CHEMICAL PROPERTIES OF ARSENIC.

Arsenic occurs in nature as a monoisotopic element, composed of one stable isotope, As. As of 2003, at least 33 radioisotopes have also been synthesized, ranging in atomic mass from 60 to 92. The most stable of these is ³³As with a half-life of 80.30 days. All other isotopes have half- lives of under one day ( Gokcen, N. A,1989).

                                                               Fig 1.1: crystal structure of arsenic

When heated in air, arsenic oxidizes to arsenic trioxide; the fumes from this reaction have an odor resembling garlic. This odor can be detected on striking arsenide minerals such as arsenopyrite with a hammer. Arsenic (and some arsenic compounds) sublimes upon heating at atmospheric pressure, converting directly to a gaseous form without an intervening liquid state at 887 K (614 °C). The triple point is 3.63 MPa and 1,090 K (820 °C). Arsenic makes arsenic acid with concentrated nitric acid, arsenious acid with dilute nitric acid, and arsenic trioxide with concentrated sulfuric acid.Arsenic compounds are used in making special types of glass, as a wood preservative and, lately, in the semiconductor galliumarsenade, which has the ability to convert electric current to laser light. Arsine gas AsH₃, has become an important dopant gas in the microchip industry, although it requires strict guidelines regarding its use because it is extremely toxic (Norman, Nicholas C 1998}.   Arsenic compounds resemble in some respects those of phosphorus which occupies the same group (column) of the periodic table. Arsenic is less commonly observed in the pentavalent state, however. The most common oxidation states for arsenic are: −3 in the arsenides, such as alloy-like intermetallic compounds, +3 in the arsenites, and +5 in the arsenates and most organoarsenic compounds. Arsenic also bonds readily to itself as seen in the square As3−4 ions in the mineral skutterudite.^[14] In the +3 oxidation state, arsenic is typically pyramidal owing to the influence of the lone pair of electrons.
Arsenic forms colorless, odorless, crystalline oxidesAs₂O₃ ("white arsenic") and As₂O₅ which are hygroscopic and readily soluble in water to form acidic solutions. Arsenic(V) acid is a weak acid. Its salts are called arsenates which are the basis of arsenic contamination of groundwater, a problem that affects many people. Synthetic arsenates include Paris Green (copper(II) acetoarsenite), calcium arsenate, and lead hydrogen arsenate. These three have been used as agriculturalinsecticides and poisons ((Martinez-Finley et al., 2012),(Madelung, Otfried 2004).

All trihalides of arsenic(III) are well known except the astatide which is unknown. Arsenic pent fluoride (AsF₅) is the only important pent halide, reflecting the lower stability of the 5+ oxidation state.  A large variety of organoarsenic compounds are known. Several were developed as chemical warfare agents during World War I, including vesicants such as lewisite and vomiting agents such as adamsite. Cacodyl acid, which is of historic and practical interest, arises from the methylation of arsenic trioxide, a reaction that has no analogy in phosphorus chemistry (Chisholm, Hugh, et al., 1911)

1.2.3 MANGANESE

Manganese is a chemical element with symbol Mn and atomic number 25. It is not found as a free element in nature; it is often found in combination with iron, and in many minerals. Manganese is a metal with important industrial metal alloy uses, particularly in stainless steels.Proposed to be an element by Carl Wilhelm Scheele in 1774, manganese was discovered by Johan Gottlieb Gahn, a Swedish chemist, by heating the mineral pyrolusite (MnO₂) in the presence of charcoal later that year. Today, most manganese is still obtained from pyrolusite, although it is usually burned in a furnace with powdered aluminum or is treated with sulfuric acid (H₂SO₄) to form manganese sulfate (MnSO₄), which is then electrolyzed. Manganese phosphating is used as a treatment for rust and corrosion prevention on steel. Depending on their oxidation state, manganese ions have various colors and are used industrially as pigments. The permanganates of alkali and alkaline earth metals are powerful oxidizers. Manganese dioxide is used as the cathode (electron acceptor) material in zinc-carbon and alkaline batteries(Lide, David R. et al, 2004.)

In biology, manganese(II) ions function as cofactors for a large variety of enzymes with many functions Manganese enzymes are particularly essential in detoxification of superoxide free radicals in organisms that must deal with elemental oxygen. Manganese also functions in the oxygen-evolving complex of photosynthetic plants. The element is a required trace mineral for all known living organisms but is a neurotoxin. In larger amounts, and apparently with far greater effectiveness through inhalation, it can cause a poisoning syndrome in mammals, with neurological damage which is sometimes irreversible ((ATSDR b,et al 2007).

1.2.4    PHYSICAL AND CHEMICAL PROPERTIES OF MANGANESE

Manganese is a pinkinsh-gray, chemically active element. It is a hard metal and is very brittle. It is hard to melt, but easily oxidized. Manganese is reactive when pure, and as a powder it will burn in oxygen, it reacts with water (it rusts like iron) and dissolves in dilute acids. Manganese is one of the most abundant metals in soils, where it occurs as oxides and hydroxides, and it cycles through its various oxidation states. Manganese occurs principally as pyrolusite (MnO₂), and to a lesser extent as rhodochrosite (MnCO₃). More than 25 million tonnes are mined every year, representing 5 million tons of the metal, and reserves are estimated to exceed 3 billion tonnes of the metal. The main mining areas for manganese ores are South Africa, Russia, Ukraine, Georgia, Gabon and Australia. Manganese is an essential element for all species. Some organisms, such as diatoms, molluscs and sponges, accumulate manganese. Fish can have up to 5 ppm and mammals up to 3 ppm in their tissue, although normally they have around 1 ppm (Rancke-Madsen, E., 1975)

Manganese metal and its common ions are paramagnetic Manganese tarnishes slowly in air and "rusts" like iron, in water containing dissolved oxygen. Naturally occurring manganese is composed of one stable isotope, Mn. Eighteen radioisotopes have been characterized, with the most stable being Mn with a half-life of 3.7 million years, Mn with a half-life of 312.3 days, and Mn with a half-life of 5.591 days. All of the remaining radioactive isotopes have half-lives that are less than three hours and the majority of these have half-lives that are less than one minute. This element also has three metal states.The most stable oxidation state for manganese is +2, which has a pale pink color, and many manganese(II) compounds are known, such as manganese(II) sulfate (MnSO₄) and manganese(II) chloride (MnCl₂) (Corathers, Lisa A., 2009)

 This oxidation state is also seen in the mineral rhodochrosite (manganese (II) carbonate). The +2 oxidation number of Mn results from removal of the two 4s electrons, leaving a "high spin" ion in which all five of the 3d orbitals contain a single electron. Absorption of visible light by this ion is accomplished only by a spin-forbidden transition in which one of the d electrons must pair with another, to give the atom a change in spin of two units.    Manganate (VI) salts can also be produced by dissolving Mn compounds, such as manganese dioxide, in molten alkali while exposed to air. Solutions of potassium permanganate were among the first stains and fixatives to be used in the preparation of biological cells and tissues for electron microscopy (Corathers, L. A.; Machamer, J. F., 2006).

1.2.6    MECHANISM OF ACTION OF ARSENIC.

Arsenite inhibits not only the formation of acetyl-CoA but also the enzyme succinic dehydrogenase. Arsenate can replace phosphate in many reactions. It is able to form Glc-6-Arsenate in vitro; therefore it has been argued that hexokinase could be inhibited. (Eventually this may be a mechanism leading to muscle weakness in chronic arsenic poisoning.) In the glyceraldehyde-3-P-dehydrogenase reaction arsenate attacks the enzyme-bound thioester. The formed 1-arseno-3-phosphoglycerate is unstable and hydrolyzes spontaneously. Thus, ATP formation in Glycolysis is inhibited while bypassing the phosphoglycerate kinase reaction. (Moreover, the formation of 2,3-bisphosphoglycerate in erythrocytes might be affected, followed by a higher oxygen affinity of hemoglobin and subsequently enhanced cyanosis) As shown by Gresser (1981), submitochondrial particles synthesize Adenosine-5’-diphosphate-arsenate from ADP and arsenate in presence of succinate. Thus, by a variety of mechanisms arsenate leads to an impairment of cell respiration and subsequently diminished ATP formation. This is consistent with observed ATP depletion of exposed cells and histopathological findings of mitochondrial and cell swelling, glycogen depletion in liver cells and fatty change in liver, heart and kidney (Hughes MF July, 2002).

Experiments demonstrated enhanced arterial thrombosis in a rat animal model, elevations of serotonin levels, thromboxane and adhesion proteins in platelets, while human platelets showed similar responses. The effect on vascular endothelium may eventually be mediated by the arsenic-induced formation of nitric oxide. It was demonstrated that +3 As concentrations substantially lower than concentrations required for inhibition of the lysosomal protease cathepsin L in B cell line TA3 were sufficient to trigger apoptosis in the same B cell line,

while the latter could be a mechanism mediating immunosuppressive effects. Another aspect is the similarity of arsenic effects to the heat shock response. Short-term arsenic exposure has effects on signal transduction inducing heat shock proteins with masses of 27, 60,70,72,90,110 kDa as well as metallotionein, ubiquitin, mitogen-activated [MAP] kinases, extracellular regulated kinase [ERK], c-jun terminal kinases [JNK] and p38. Via JNK and p38 it activates c-fos, c-jun and egr-1 which are usually activated by growth factors and cytokines. The effects are largely dependent on the dosing regime and may be as well inversed (Gresser MJ June, 1981).

As shown by some experiments reviewed by Del Razo (2001), ROS induced by low levels of inorganic arsenic increase the transcription and the activity of the activator protein 1 (AP-1) and the nuclear factor-κB (NF-κB) (maybe enhanced by elevated MAPK levels), which results in c-fos/c-jun activation, over-secretion of pro-inflammatory and growth promoting cytokines stimulating cell proliferation. Germolec et al. (1996) found an increased cytokine expression and cell proliferation in skin biopsies from individuals chronically exposed to arsenic-contaminated drinking water.

Increased AP-1 and NF-κB obviously also result in an up-regulation of mdm2 protein, which decreases p53 protein levels.^] Thus, taking into account p53’s function, a lack of it could cause a faster accumulation of mutations contributing to carcinogenesis. However, high levels of inorganic arsenic inhibit NF-κB activation and cell proliferation. An experiment of Hu et al. (2002) demonstrated increased binding activity of AP-1 and NF-κB after acute (24 h) exposure to +3 sodium arsenite, whereas long-term exposure (10–12 weeks) yielded the opposite result. The authors conclude that the former may be interpreted as a defense response while the latter could lead to carcinogenesis. As the contradicting findings and connected mechanistic hypotheses indicate, there is a difference in acute and chronic effects of arsenic on signal transduction which is not clearly understood yet (Hu Y, Su L, Snow ET September, 1998).

1.2.7 ABSORPTION AND METABOLISM OF ARSENIC

The major site of absorption is the small intestine by an electrogenic process involving a proton (H+) gradient. The optimal pH for arsenic absorption is 5.0,38 though in the milieu of the small bowel the pH is approximately 7.0 due to pancreatic bicarbonate secretion. The absorbed arsenic undergoes hepatic biomethylation to form monomethylarsonic acid and dimethylarsinic acid that form monomethylarsonic acid and dimethylarsinic acid that are less toxic but not completely innocuous.  About 50% of the ingested dose may be eliminated in the urine in three to five days. Dimethylarsinic acid is the dominant urinary metabolite (60%–70%) compared with monomethylarsonic acid. A small amount of inorganic arsenic is also excreted small amount of inorganic arsenic is also excreted unchanged. After acute poisoning electrothermal atomic absorption spectrometry studies show that the highest concentration of arsenic is in the kidneys and liver (Vahter M, Concha G July 2001).

In chronic arsenic ingestion, arsenic accumulates in the liver, kidneys, heart, and lungs and smaller amounts in the muscles, nervous system, gastrointestinal tract, and spleen. Though most arsenic is cleared from these sites, residual amounts remain in the keratin-rich tissues, nails, hair, and skin. After about two weeks of ingestion, arsenic is deposited (Styblo M, Thomas DJ April, 2001).

1.2.8 TOXICITY OF MANGANESE

Manganism or manganese poisoning is a toxic condition resulting from chronic exposure to manganese. It was first identified in 1837 by James Couper.Chronic exposure to excessive manganese levels can lead to a variety of psychiatric and motor disturbances, termed manganism. Generally, exposure to ambient manganese air concentrations in excess of 5 micrograms Mn/m3 can lead to Mn-induced symptoms(Kulig et al., 1996).

In initial stages of manganism, neurological symptoms consist of reduced response speed, irritability, mood changes, and compulsive behaviors. Upon protracted exposure symptoms are more prominent and resemble those of idiopathicParkinson's disease, as which it is often misdiagnosed, although there are particular differences in both the symptoms (nature of tremors, for example), response to drugs such as levodopa, and affected portion of the basal ganglia. Symptoms are also similar to Lou Gehrig's disease and multiple sclerosis(Santamaria, 2008).

Excess manganese interferes with the absorption of dietary iron. Long-term exposure to excess levels may result in iron-deficiency anemia. Increased manganese intake impairs the activity of coppermetallo-enzymes. Manganese overload is generally due to industrial pollution. Workers in the manganese processing industry are most at risk. Well water rich in manganese can be the cause of excessive manganese intake and can increase bacterial growth in water. Manganese poisoning has been found among workers in the battery manufacturing industry (Stansbie, John Henry,2007).
Symptoms of toxicity mimic those of Parkinson's disease (tremors, stiff muscles) and excessive manganese intake can cause hypertension in patients older than 40. Significant rises in manganese concentrations have been found in patients with severe hepatitis and posthepaticcirrhosis, in dialysis patients and in patients suffering heart attacks.

Manganese influences the copper and ironmetabolism and estrogen therapy may raise serum manganese concentration, whereas glucosteroids alter the manganese distribution in the body. Calcium deficiency increases manganese absorption. Elevated calcium and/or phosphorus intake suppress the body's ability to absorb manganese, while an increase in Vitamin C improves cellular exchange. Manganese overload is generally due to industrial pollution. Workers in the manganese processing industry are most at risk. Drinking water should be analyzed when manganese toxicity is suspected. Long term parenteral nutrition has been associated with high blood concentrations of manganese in children who displayed symptoms of toxicity (Silva Avil et al,2013).
Dark hair dyes can contain manganese and thus falsely elevate hair levels. In the case of extremely high manganese levels obtained from scalp hair, pubic hair should be tested as a control.Manganism is a biphasic disorder. In its early stages, an intoxicated person may experience depression, mood swings, compulsive behaviors, and psychosis. Early neurological symptoms give way to late-stage manganism, which resembles Parkinson's disease. Symptoms include weakness, monotone and slowed speech, an expressionless face, tremor, forward-leaning gait, inability to walk backwards without falling, rigidity, and general problems with dexterity, gait and balance. Unlike Parkinson's disease, manganism is not associated with loss of smell and patients are typically unresponsive to treatment with L-DOPA. Symptoms of late-stage manganism become more severe over time even if the source of exposure is removed and brain manganese levels return to normal( Finley, John Weldon; Davis, Cindy D. ,1999).

1.3.0       EPIDERMOLOGY OF ARSENIC AND MANGANESE WORLDWIDE

Establishment of the maximum contaminant level that regulates the concentration of arsenic in public water supplies in the United States was a protracted process. The Public Health Service (PHS) set an interim standard of 50 ug/I in 1942 and stated that the goal should be 10 ug/L in 1962, but it was another forty years before the U.S. Environmental Protection Agency actually lowered the standard to 10 ug/1. Despite extensive epidemiological evidence of significant cancer risks accumulated over many years, the US flip-flopped on the drinking water standard before and after the transition from the Clinton to the Bush Administrations. One problem is that regulators, and many scientists, having learned the terms "confounding" and "exposure misclassification", appear to be more comfortable with the results of experimental animal studies than human epidemiological studies. In the case of arsenic, there are clear increased risks of human cancer once concentrations reach 200ug/L in drinking water, whereas there is little response in standard animal bioassays, even at concentrations of 50,000ug/L and above. Furthermore, at concentrations above 500ug/L, the human risks are extraordinarily high, with one in ten exposed persons dying from arsenic-caused cancers. Such contrasts in cancer risks between animals and humans are unprecedented. Furthermore, the lung may be the main site of long-term human health effects from ingestion of arsenic in water (which is hard to swallow), and epidemiological data suggest that the risk from arsenic inhalation may be equivalent to that from ingestion (also hard to swallow). There are important lessons to be learned from the history of arsenic drinking water regulations and a lot more yet to learn from epidemiological studies of the health effects of human exposure to arsenic.

1.3.1    EPIDIDYMIS

The epididymis   is a tube that connects a testicle to a vas deferens in the male reproductive system. It is present in all male reptiles, birds, and mammals. It is a single, narrow, tightly-coiled tube (in adult humans, six to seven meters in length connecting the efferent ducts from the rear of each testicle to its vas deferens.

                                                   Fig1.2: structure of epididymis

The epididymis can be divided into three main regions:

• The head: The head of the epididymis receives spermatozoa via the efferent ducts of the mediastinium of the testis. It is characterized histologically by a thin myoepithelium. The concentration of the sperm here is dilute.
• The body
• The tail :This has a thicker myoepithelium than the head region, as it is involved in absorbing fluid to make the sperm more concentrated.

In reptiles, there is an additional canal between the testis and the head of the epididymis and which receives the various efferent ducts. This is, however, absent in all birds and mammals

FUNCTION

Role in storage of sperm and ejaculant

Spermatozoa formed in the testis enter the caput epididymis, progress to the corpus, and finally reach the cauda region, where they are stored. Sperm entering the caput epididymis are incomplete—they lack the ability to swim forward (motility) and to fertilize an egg. It stores the sperm for 2–3 months. During their transit in the epididymis, sperm undergo maturation processes necessary for them to acquire these functions. Final maturation is completed in the female reproductive tract.

During ejaculation, sperm flow from the lower portion of the epididymis (which functions as a storage reservoir). They have not been activated by products from the prostate gland, and they are unable to swim, but are transported via the peristaltic action of muscle layers within the vas deferens, and are mixed with the diluting fluids of the seminal vesicles and other accessory glands prior to ejaculation (forming semen)

The epithelial cells of the epididymis possess numerous apical modifications that are often referred to as stereocilia, as under the light microscope they look like cilia. However, as electron microscopy has revealed them to be structurally and functionally more similar to microvilli, some now refer to them as stereovilli.

1.4.0 FREE RADICAL AND OXIDATIVE DAMAGE

The human body is constantly under attack from free radicals. A free radical is any chemical specie capable of independent   existences and possessing one or more unpaired electrons, an unpaired electron being one that is alone in an orbital. Free radicals are generated by biological chemical redox reactions that occur as normal body process. They are highly unstable and have electrons that readily pair with organic substrates. Free radicals are generated when cells use oxygen to generate energy as a result of ATP production by the mitochondria. Exposure to environmental factors like heavy metals in which arsenic and manganese belong to ,ultra-violate light, cigarette smoke , environmental pollutants and gamma radiation (Demasi et al., 1996; Emanuelliet al., 2003; Juknatet al., 1995).

Free radicals react with organic substrates such as lipids, proteins and DNA. Oxidation of these molecules can damage them, disturbing normal functions and may contribute to a variety of disease state like cancer, apoptosis etc. (Aroumaet al., 1998)

1.4.1     BASIC TYPES OF DAMAGES CAUSED BY FREE RADICALS

Ø  Lipid peroxidation: free radicals initiate damage to fat components in the body causing them to turn rancid and release more radicals.

Ø  Membrane damage: the integrity of the cell membrane is damaged due to reaction of free radicals. This in turn interfers with the cell’s ability to take in nutrients and expel waste.

Ø  Lysosomal damage: the lysosomal membrane containing hydrolytic enzymes is ruptured as a result of free radicals, hence this enzyme spills out of the lysosome into the cell causing digestion of critical compounds and molecules in the cell.

Ø  Cross linking: free radicals reactions cause protein or DNA to fuse together (Brillaet al., 1995).

1.4.2 FORMATION OF FREE RADICALS

Interest in free radicals began with the work of Moses Gomberg (1), who in 1900 demonstrated the existence of the triphenylmethyl radical (Ph3C·). A free radical is any chemical species (capable of independent existence) that possesses one or more unpaired electrons, an unpaired electron being one that is alone in an orbital.  Atoms are mostly stable in their ground state. An atom is considered to be in their ground state when every electron in the outermost shell has a complimentary electron that spins in the opposite direction. A free radical is easily formed when a covalent bond splits and one electron remains with each newly formed atom. Free radicals are incapable of existing alone and readily look for an atom or molecule to extract electrons from, to complete their lone orbitals. The following literature review concentrates on radicals with an oxygen center; these are referred to as reactive oxygen species (ROS). ROS contain two unpaired electrons in their outermost shell. Once radicals form, they can react either with another radical or with another molecule by various interactions. This leads to formation of a chain of free radicals (Juknat et al., 1995). Free radicals can be formed from various reactions in the body. Some of the include;

1. Generation of ATP (universal energy currency) from ADP in the mitochondria: oxidative phosphorylation

2. Detoxification of xenobiotics by Cytochrome P450 (oxidizing enzymes)

3. Apoptosis of effete or defective cells

4. Killing of micro-organisms and cancer cells by macrophages and cytotoxic lymphocytes

5. Oxygenases (eg. COX: cyclo-oxygenases, LOX: lipoxygenase) for the generation of prostaglandins and leukotrienes, which have many regulatory functions.

In the electron transport chain, oxygen acts as the electron acceptor. This literature suggests that anywhere from 2 to 5 percent of total oxygen intake during both rest and exercise have the ability to form highly damaging superoxide radical via electron escape. During exercise oxygen consumption is increased and also electron escape from the electron transport chain is enhanced about 10-20 folds.

Types Of Free Radicals

The simplest form of free radical is the hydrogen atom which consists of one electron. However this study is based mostly on the reactive oxygen species which contain unpaired electro on their outermost shell. Examples of free radicals include;hydroxyl (OH·), superoxide (O2·−) nitric oxide (NO·), and peroxyl (RO2·). Peroxynitrite (ONOO−), hypochlorous acid (HOCl), Hydrogen peroxide (H2O2), singlet oxygen 1Δg (often written as 1O2), and ozone (O3) (often written as 1O2), and ozone (O3) are not free radicals but can easily lead to free-radical reactions in living organisms.

1.4.3          REACTIVE OXYGEN SPECIES

Reactive oxygen species have long been known to be a component of the killing response of immune cells to a microbial invasion. Recent studies indicate that ROS play a key role as a messenger in normal cell transduction and cell signaling. Here we briefly describe the biology behind some of these molecules and means of their detection. ROS is a phrase used to describe a number of reactive molecules and free radicals derived from molecular oxygen. The production of oxygen based radicals is general for all aerobic species. These molecules are produced as byproducts during biological reactions like mitochondrial electron transport chain reaction or by oxireductase enzymes and metal catalyzed oxidation and have deleterious effects on the body. It was originally thought that only phagocytic cells were responsible for ROS as part of their defense mechanism. Recent studies have however demonstrates that ROS play a role in the following; cell signaling, including apoptosis, gene expression and activation of cell signaling cascades (Orreniuset al)

Types of Free Reactive Oxygen Species

·         superoxide (O^2-) anion

·          hydrogen peroxide (H₂O₂)

·         peroxyl (ROO^-) radical

·         the very reactive hydroxyl (OH^-)

·         nitricoxide (NO^.)

Hydroxyl radical

Hydroxyl radical (OH) is the most damaging free radical and has devastating effect on the body. It is a third generation radical and is derived from H202 hydrogen peroxide which is derived from superoxide radical through the enzyme superoxide dismutase.  Hydrogen peroxide is reduced to hydroxyl radical by the enzymes glutathione peroxidase and catalase in the presence of transition metals like iron and copper. The dangers of OH have been highlighted by Dr Reiter as follows; ‘if the function of radical is to destroy molecules and tissues, then the OH is the radical’s radical.  It reacts at diffusion rate with virtually any molecule found in its path including macromolecules like DNA, membrane lipids, proteins and carbohydrates. In the DNA, it can induce strand breaks as well as chemical changes in the purine and pyrimidine bases and can cause proteins to lose their efficiency.

Peroxyl radicals

This contains a superoxide molecule with the chemical formula (O2^·−). The systematic name of the anion is (1^-). Superoxide anion is particularly important as the product of one electron reduction of dioxygen which occurs widely in nature. With one unpaired electron, the superoxide ion is a free radical.

Ozone

Ozone is a powerful oxidant and has many industrial and consumer applications related to oxidation. This same high oxidizing potential causes it ozone to damage mucus and respiratory tissues in animals and also tissues in plants above concentration of about 100 parts per billion. However, the so called ozone layer which is a portion of the stratosphere with a higher concentration of ozone is beneficial preventing damaging ultra violet light from reaching the earth”s surface, to the benefit of both man and plants.

Superoxide anion

Superoxide is biologically quite toxic and is developed by the immune system to kill invading microbes.  In phagocytes, it is produced in large quantities by the enzyme NADPH oxidase for use in oxygen –dependent killing mechanisms of invading pathogens.

Hydrogen peroxide

It is a reactive metabolic by product that is a key regulator in a number of oxidative stress related states. Functioning through NFKB and other factors, its mediated pathway has been linked to asthma, atherosclerosis, diabetic vasculopathy, osteosporosis, a number of neurodegenerative diseases and down’syndrome. It is generated invivo by the mitochondrial respiratory chain as well as by a range of oxidase enzymes. It is eliminated via the actions of catalases and peroxidases.

Hypochlorous acid:

It has simply been thought of as a transient by product in the ubiquitous chlorine chemical family. However, it has been shown to carry fewer negative hydroxides. HOCL as a stand-alone chemical, separate from chlorine has not been available in the market until now.

Measurement of free radicals:

Free radicals are difficult to trap and measure, because they have short half-life. However multiple methods have been devised for their measurement. Radicals can be measured using electron spin resonance and spin trapping methods where exogenoius compounds that have high affinity for the radicals are used. The compound and the radical together form a stable entity that can be measured. This method is however not so accurate. Another method is using free radical markers. These markers of oxidative stress are measured using a variety of assays which are decribed below. When a fatty acid is peroxidizes, it is broken down into aldehydes which are excreted. Aldehydes such as thiobarbutric acid reacting substances (TBARS) have been widely accepted as a general marker of free radical production egmalonaldehyde (MDA). The TBA test has been questioned because of its lack of specificity, sensitivity and reproducibility. The uses of liquid chromatography instead of spectrophotometry techniques help reduce the error. Lastly conjugated dienes (CD) are often measured as indicators of free radicals Oxidation of unsaturated fatty acids result in the production of CD. These are measured and provide a marker of the early stages of lipid peroxidation.  A newly developed method uses monoclonal antibodies and may prove to be the most accurate method (Dillard et al,Kanter et al)

Physiological effects:

Usually, the body is able to handle free radical production using antioxidants, however, during increased oxygen flux; free radical production may exceed that of removal resulting in lipid peroxidation. Free radicals have been implicated in the etiology of diseases like cardiovascular diseases, cancer, Alzheimer diseases and Parkinson’sdisease.  The literature review will however review lipid peroxidation and its driving force.

Importance of free radicals;

This review has based on the negative effects of free radicals; however they might actually play some important roles in the body. Free radicals are naturally produced by some cells in the body like the phagocytes as a host defense mechanism. The immune system is the main body system that utilizes free radicals. Foreign invaders or damaged tissue is marked with free radicals by the immune system. This allows for determination of which tissue need to be removed from the body. Because of this some question that the need for antioxiudant supplementation as they believe it can actually decrease the effectiveness of the immune system.

1.4.4 LIPID PEROXIDATION.

Lipid peroxidation is one of the most widely used indicators of free radical formation, a key indicator of oxidative stress. Unsaturated fatty acids are present im the biological membrane are are easy targets of free radicals. This reaction occurs as a chain of reaction where a free radical will capture a hydrogen molecule from an unsaturated fatty acid to form water. It contributes to the development of cardiovascular diseases, such as preeclampsia and atherosclerosis, and the end-products of this process [particularly cytotoxic aldehydes, such as malondialdehyde  (MDA) and 4-hydroxynonenal (HNE)] can cause damage to proteins and to DNA. Peroxidation causes impairment of biological membrane functioning, e.g., decreases fluidity, inactivates membrane-bound enzymes and receptors, and may change nonspecific calcium ion permeability. It can be initiated by a free radical that has sufficient reactivity to abstract a hydrogen atom from a poly unsaturated fatty acid (PUFA) side chain in membrane lipids or plasma lipoprotein particles.                                                                                                                                                                             Lipid peroxidation is a free radical mediated process.  Initiation of a peroxidative sequence is due to the attack by any species, which can abstract a hydrogen atom from a methylene group (CH2), leaving behind an unpaired electron on the carbon atom (•CH). The resultant carbon radical is stabilized by molecular rearrangement to produce a conjugated diene, which then can react with an oxygen molecule to give a lipid peroxyl radical (LOO•). These radicals can further abstract hydrogen atoms from other lipid molecules to form lipid hydroperoxides (LOOH) and at the same time propagate LP further. The peroxidation reaction can be terminated by a number of reactions. The major one involves the reaction of LOO• or lipid radical (L•) with a molecule of antioxidant such as vitamin E or α-tocopherol (α-TOH) forming more stable tocopherolphenoxyl radical that is not involved in further chain reactions. This can be ‘recycled’ by other cellular antioxidants such as vitamin C or GSH. This can be ‘recycled’ by other cellular chain reactions.

LH + •OH → L• + H2O

L• + O2 → LOO

LOO• + LH → L• + LOOH

LOO• + α-TOH → LOOH + α-TO•

The process of LP, gives rise to many products of toxicological interest like malondialdehyde (MDA), 4- hydroxynonenal (4-HNE) and various 2-alkenals. Isoprostanes are unique products of lipid peroxidation of arachidonic acid and recently tests such as mass spectrometry and ELISA-assay kits are available to detect isoprostanes (Yoshikawa et al. 2000). TBARS have been shown to react with these aldehyde products to form coloured complexes which absorb radiation at specific wavelengths. This reaction has been used to access the extent of lipid peroxidation.

1.4.5 OXIDATIVE STRESS

The relation between free radicals and disease can be explained by the concept of ‘oxidative stress’ elaborated by Sies (1986).12 In a normal healthy human body, the generation of pro-oxidants in the form of ROS and RNS are effectively kept in check by the various levels of antioxidant defense. However, when it gets exposed to adverse physicochemical, environmental or pathological agents such as atmospheric pollutants, cigarette smoking, ultraviolet rays, radiation, toxic chemicals, overnutrition and advanced glycation end products  (AGEs) in diabetes, this delicately maintained balance is shifted in favor of pro-oxidants resulting in ‘oxidative stress’. It has been implicated in the etiology of several (>100) of human diseases and in the process of ageing.

The damaging of the DNA which is caused by carcinogenic ionizing radiation is known to be mediated through the mutagenic effects of hydroxyl radicals and due to cancer being strongly correlated with age, normal aging which is attributed to the accumulation of unrepaired mutagenic DNA lesions and oxidative stress has been implicated in the free radical theory of aging (Ames et al., 1983) and (Beckman et al., 1980).

1.4.6 ANTIOXIDANTS

Exposure to free radicals from a variety of sources has led organisms to develop a series of defence mechanisms (Cadenas, 1997). Defence mechanisms against free radical-induced oxidative stress involve: (i) preventative mechanisms, (ii) repair mechanisms, (iii) physical

defences, and (iv) antioxidant defences. Enzymatic antioxidant defences include superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). Non-enzymatic antioxidants are represented by ascorbic acid (Vitamin C), _-tocopherol (Vitamin E), glutathione (GSH), carotenoids, flavonoids, and other antioxidants. Under normal conditions, there is a balance between both the activities and the intracellular levels of these antioxidants. This balance is essential for the survival of organisms and their health (Brillaet al., 1995)

Antioxidant Defenses

Antioxidant means against. Antioxidants work in the body to reduce and prevent damage of cellular components by free radicals. They are effective because they are willing to donate their electrons to free radicals and in this way prevent them from extracting electrons from the cellular components. Antioxidants are naturally built to accommodate this electron change within them and though they become free radical by definition after releasing this electron, they are not harmful to the body. Antioxidants are manufactured within the body and can also be gotten from food like fruits, vegetables, seeds, nuts, meats and oil. There are two lines of antioxidant defense in the body. The first line found in the fat soluble vitamin E, beta –carotene, and coenzyme Q. of these, vitamin E is considered the most potent chain breaking antioxidant.  Inside the cell, water soluble scavangers are present. These include vitamin, glutathione peroxidase, superoxide dismutase and catalase. These are discussed below.

Glutathione

Glutathione is a cysteine containing peptide that is found in most forms af aerobic life and is not required in diet but is synthesized within the cell from constituent amino acids. It is a tripeptide of glutamate, cysteine and glycine containing an unusual peptide linkeage. The existence of the bond between them prevents hydrolyzation by most peptidase. Its antioxidant property is due to the fact that the cysteine moiety is a reducing agent and can be reversibly oxidized and reduced. It is maintained in the reduced form in the cell.  It is present in high concentration in the cell. A major function of GSH as an antioxiudant is the reduction of H202 and other peroxidases by a reaction catalyzed by glutathione peroxidase. It also takes part in non-enzymatic reductions. The oxidized form of glutathione is (GSSG) and is converted back to GSH by an enzyme glutathione reductase in an NADPH dependent manner.

Glutathione peroxidase contains four selenium ions and is found in different fraction of cells and tissues in the body.  It is the only known enzyme that requires Se for its activity in the body and this may be related to the current interest in the dietary supplements of Se to prevent cancer (Anderson et al., 1998)

Glutathione S- Transferases

GST are a major group of detoxification enzymes possessed by all eukaryotic species and can be cytosolic or membrane bound.  The cytosolic enzymes are encoded by five distantly related gene families whereas the membrane bound enzymes, microsomal GST and leukotriene C4 synthetase are encoded by single genes and both have arisen separately from soluble GST. GST has been considered among several others to contribute to the phase 2 biotransformation of drugs. They do this by conjugating these compounds with reduced glutathione to facilitate dissolution in the aqueous cellular and extracellular media and from there out of the body.

Reduced Glutathione (GSH)

GSH protect the cell from free radicals by oxidation. It can only function in its reduced state so that it can readily neutralize free radicals by bonding with them. It bonds and converts to its oxidized form and is converted back to its reduced state by glutathione reductase. The ratio of its oxidized form to its reduced form can be used to measure cellular toxicity. 90 percent of cellular GSH is in its reduced form.

Vitamin C and E

Vitamin c isa monosaccharide found in both plants and animals. It is gotten from dietary intake. Most animals produce it in their body and do not need to take them in food. It’s also called ascorbic acid. It is required for conversion of procollagen to collagen. It is maintained in its reduced form by its reaction with glutathione which can be catalyzed by protein disulfide isomerase and glutaredoxins. It is a redox catalyst and can reduce and therefore neutralize free radicals. Vitamin e also known as alpha tocopherol is a fat soluble vitamin found in vegetables oilseed, fishoil, whole grains, apicrotsetc its biological activity is to maintain polyunsaturated fatty acids and membrane qualities, it functions as a peroxyl scavenger that terminates chain reactions.  There are important differences between various vitamin E forms with respect to their antioxidant activity when measured invitro.

Catalase

It is a common compound found in all living organisms that are exposed to oxygen and catalyzes the decomposition of hydrogen peroxide to water and oxygen. It is a tetramer of four polypepetides chains, each over 500 amino-acid long (Boon et al., 2007). It contains four porphyrinheme irons.

H₂O₂ →  H₂O + 1/2O₂

The true biological significance of catalase is not always straightforward to assess: mice genetically engineered to lack catalase are phenotypically normal, indicating that this enzyme is dispensable in animals under normal condition (HoYSet al., 2004). A catalase deficiency may increase the likelihood of developing Type2 Diabetes (Lazlo et al., 2008).  Some human beings have very low levels of catalase (acatalasia) yet show few ill effects. It is likely that the predominant scavengers of H2O2 in normal mammalian cells are peroxiredoxins rather than catalase.  Human catalase works at an optimum temperature 0f 37c.  The catalase test is also used by microbiologist to identify the species of bacteria. The presence of catalase enzyme in the test isolate is detected using hydrogen peroxide. If the bacteria possess catalase (i.e., are catalase positive), when a small amount of bacterial isolate is added to hydrogen peroxide, bubbles of oxygen are observed.

Catalase can also catalyze the oxidation, by hydrogen peroxide by hydrogen peroxide of various metabolites and toxins, including formaldehyde, formic acid, phenols, acetaldehyde and alcohols. It does so according to the following reaction;

H₂O₂+H₂R→2H₂O+R               

While the complete mechanism of catalase is not currently known (boon et al., 2007), the reaction is believed to occur in two stages:

 (IV)-E+O2 (. +)

1.5 AIM AND OBJECTIVES

The administration of the toxicants would last for two weeks after which we are to sacrifice and extract the organs needed and make experimental observation by comparison to control. Out of the sixty four animals bought, 31 would be sacrificed after first two weeks of treatment, the remaining would be left without treatment with toxicant and then sacrificed after another two weeks and experiment carried out on extracted organs.

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