Effect of Aflatoxin and Protein Malnourishment on the Hepatic Lipids and Oxidative Stress

                                              

CHAPTER ONE

1. BACKGROUND

Exposure to aflatoxin or its metabolites have been shown to play an important role in oxidative stress which may result in aflatoxicosis. Aflatoxin such as aflatoxin B1 is regarded as a mycotoxin that can cause contamination in food (Fatima et al., 2015).

Aflatoxins are poisonous by-product of fungi mainly Aspergillus which are Aspergillus parasiticus and Aspergillus flavus. It can be found in various food crops such as corn, millet rice, groundnut, sorghum and others (Komsky- Elbaz, 2018). The main four types of aflatoxins are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) (Wu et al., 2016). Others include aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2) which are metabolites of AFB1. Aflatoxin B1 (AFB1) can cause oxidative DNA damage in rat liver, whereby hydroxyl radicals may be involved as the initiation species (Gradelet et al., 1998).

Protein malnourishment has been a form of malnutrition that occurs when there is an imbalance between protein intake and the amount that is required by the body for optimal growth and development (Fock et al., 2010; Bossola, 2015). Protein malnourishment produces oxidative stress in a study where the effect of protein –free diet is noted for 5 days on mouse liver (Veronica et al., 2015), indicating that low- protein diet can induce oxidative stress.

Oxidative stress is said to be an imbalance between the productions of free radicals (reacting oxidative species) generated during oxidative metabolism and antioxidants that is responsible for scavenging the free radicals generated.

Fig 1.1 Illustration of oxidative stress (Kohen and Niskar, 2002; Schafer and Buettner, 2001)

Free radicals are molecules that contain oxygen with one or more unpaired electrons, thus enabling it react with other molecules (Ananya, 2017). Excess free radicals may result in oxidation of critical biological molecules, lipid peroxidation production, damage of certain protein and also involve in DNA strand breaks (Halliwell, 1992).

Fig1.2 Pathway for free radicals generation (Winterbourn, 2008)

Oxidative damage occurs in a cell or tissue when the ratio of reactive oxygen species such as hydrogen peroxide which is generated to that of the antioxidant present; however, the result of the decrease in the non-enzymatic antioxidant (vitamin C, vitamin E and glutathione) and enzymatic antioxidants (superoxide dismutase, catalase, glutathione peroxidase) levels can also lead to oxidative stress. Oxidative stress can also lead to cell damage (Sies, 1991). Oxidative damage is a cause of harm to the DNA causing trauma and it has reported to be linked to cancer.

Protein malnourishment and aflatoxin have been reported to cause damage to the hepatic cells. Both can also alter the liver biochemical characteristics and histology (Cabarello et al., 2011).

1. RESEARCH PROBLEM

Protein malnourishment and aflatoxin from several discoveries have been found to cause oxidative stress and also cause oxidative damage.

The ingestion of aflatoxin contaminated food is associated with liver diseases (Sekena, 2001).Protein malnourishment can alter the biochemical characteristics of liver (Caballero et al., 2011; Ronchi et al., 2010; Ronchi et al., 2011). Therefore, aflatoxin and protein malnourishment can induce liver cancer.

2. JUSTIFICATION OF STUDY

The possible role of aflatoxin is its carcinogenic effects on human health, there can also be other or additional health risks caused by aflatoxin when uncontrolled. Therefore, it would of great importance to have an effective study on the possible effects of aflatoxin on human health in developing countries (Williams et al., 2004).

It has been reported that the effects caused by aflatoxin are similar such as fatty liver, immunosuppression (Hamilton, 1997). This study would show the relationship between the effects aflatoxin and protein malnourishment has on human health.

3. AIMS AND OBJECTIVES

Aim: To study the effect of aflatoxin and protein malnourishment on the hepatic lipids and oxidative stress in albino rats.

The objectives of this study include:

• Effects of aflatoxin B1 and protein malnourishment on the hepatic oxidative damage.
• Effects of aflatoxin B1 and protein malnourishment on the hepatic and circulating lipids.

 

 

 

 

 

 

 

 

 

 

CHAPTER TWO

2.1 MYCOTOXIN

Mycotoxin was derived from mycotoxicosis; a termed used in 1955 to describe diseases of animals caused by toxics metabolic by-products of certain fungi (Hermann, 2002) such as mushroom, moulds and yeast. Mycotoxins are secondary metabolites that are toxic and they are produced by filamentous fungi. According to Food and Agriculture Organization (FAO) of United Nations, 25% of the world’s crops are contaminated with mycotoxins either during growth or storage (Wu, 2007).

Mycotoxins are secondary metabolites that are harmful and are produced from flamentous fungi within the genera of Aspergilus, Fusarium and Penicillium (Reddy et al., 2010).  The major factors that cause the occurrence of mycotoxins at pre-harvest and post-harvest stages include high moisture and temperature (Ayckek et al., 2005). Mycotoxin has become a major challenge in tropical and sub-tropical areas under some factors such as high temperature, relative humidity, poor storage conditions and pest damage.

Fig.2.2 Prevention and decontamination process of mycotoxin (Pankaj et al., 2018)

Aflatoxin, Fumonisins, zearelenone, ochratoxin and deoxynivalenol are the major five  groups of mycotoxins that are toxic to mammal (Karlovsky et al., 2016).It has been known that mycotoxins exhibit carcinogenic, mutagenic and genotoxic properties (Pankaj et al., 2018). Diversification in the structure of mycotoxins results in different adverse health effects. For instance, ochratoxin can affect protein synthesis and inhibit production of adenosine triphosphate (ATP) (Jard et al., 2011; Parent-Massin, 2004).

Fig.2.3 Structures of mycotoxin group (Eivazzadeh-Keihan et al., 2017)

2.1.1 AFLATOXIN

2.1.1.1 HISTORY

The name ‘aflatoxin’ was named after their fungal source. The discovery of aflatoxins was in 1960 which resulted from an outbreak of mysterious disease later known as ‘Turkey X’ disease which 100, 000 Turkey poults in England were killed. It was later established that Peanut from Brazil was the cause and later discovered that Fungus produced a toxin that brought about the spoilage (Ji Pitt, 2014). The main four types of aflatoxin are Aflatoxins exist as aflatoxin A1, aflatoxin A2, aflatoxin B1, aflatoxin G1and aflatoxin G2. Other metabolites are aflatoxin M1 and aflatoxin M2.

Fig.2.1.1 Areas and population at risk of chronic exposure to aflatoxin (Williams et al., 2004)

A group of mycotoxins known as aflatoxin are produced in feeds and foods mainly by Aspergillus flavus and Aspergillus parasiticus (Ji Pitt, 2014), they are well known human substances. Aflatoxins are compounds with low molecular weight produced by Aspergillus flavus and Aspergillus parasiticus (Dai et al., 2017; Kew, 2013).

Aflatoxin contaminates food production at different stages; the pre-harvest stage and post-harvest (storage stage) (Limura et al., 2017; Shuaib et al., 2010). According to US Foods and Drug Administration, the allowed amount of aflatoxin in human food is 0.01-0.1micrometre and in animal feed should have between 1micrometre (Williams et al., 2004).

Aflatoxin can be stored in the body and thus, found in the tissues such as liver, muscles and animal product such as milk and eggs (Fan et al., 2015; Yuan et al., 2016; Monson et al., 2016).

2.1.2 SOURCES OF AFLATOXIN

Aflatoxins are mainly produced by Aspergillus species such as the Aspergillus flavus and Aspergillus parasiticus, which are common in the tropical region (Angele et al., 2010). Various food products where aflatoxin can be found are maize, sorghum, millet, rice and wheat. Aflatoxin can be found in edible tissues such as liver, muscle and eggs (Yuan et al., 2016), also in maternal breast milk and cord blood (Shuaib et al., 2010).

The major factors in mould infestation and production of toxin include insect damage, water stress and high temperature stress. The minimum, maximum and optimum condition required for the production of aflatoxin has been reported to be (12-27) and (40-42) degree Celsius

2.1.3 STRUCTURES OF AFLATOXIN

Aflatoxins belong to closely related difuranocoumarin compounds (Eaton and Evan, 1994) derived from polyketide pathway (Vederas and Nkashima, 1980). In 1964, Dicken postulated that the activity of toxin was as a result of the unsaturated sigma- lactone. Aflatoxins B belong to pentanone chemical species, and their characteristics are based on the fusion of a cyclopentanone ring to the lactone ring of the coumarin structure while aflatoxins G belong to lactone chemical species with an additional fused lactone ring.

Both Aflatoxins B and G group have an unsaturated bond at the 8, 9 position of the terminal furan ring (Kamla-Raj, 2004).

Fig.2.1.3  Chemical structures of Aflatoxin (Antonello and Alberto, 2013)

2.1. 4 TYPES OF AFLATOXIN

The four main types are classified in the group 1 as Human carcinogens proposed by International Agency for Research and Cancer (IARC, 2002). They include:

Aflatoxin B1 (AFB1), aflatoxin B2(AFB2), aflatoxin G1(AFG1), aflatoxin G2(AFG2) (Mc Millian et al., 2018). Others include aflatoxin M1(AFM1) and aflatoxin M2 (AFM2), they are found in dairy products. AFM1 and AFM2 are the metabolites of AFB1 and AFB2 respectively (Oveisi et al., 2007; Sadeghi et al., 2009).

AFLATOXIN B: This exists in two forms; Aflatoxins B1 and B2. They are named after the blue colour they fluoresce under ultraviolet light.

Aflatoxin B1 is termed as a carcinogenic compound (Cavajal-Moreno, 2015). It is the most toxic and commonly occurring type of aflatoxin (Pitt et al., 2012; Wild et al., 2015) and it is classified as group 1 human carcinogen. AFB1 has been reported to exert its action on cell-mediated immunity by the reduction of the number of circulating lymphocytes. Thus, it is immunotoxic (Hinton et al., 2003).

AFB1 is known to be an endocrine disruptor as it is involved in affecting cytochrome P450 enzymes in steroid synthesis (Storvik et al., 2011) and it is metabolised into an aflatoxin 8,9-exo-epoxide. AFB1is associated with some human health factors that can help in cancer development including Hepatitis B virus infection, nutritional status, sex, age and the amount of aflatoxins exposure (Quershi et al., 2014; Wild and Montesano, 2009).

Moreover, AFB1 have the ability to modify macrophage (important component of system responsible for stimulating innate immune responses and engulfing microbes) function (Flannagan et al., 2009).

One main derivative form of aflatoxin B1 is aflatoxin M1. Aflatoxin M1 is mostly found in urine and mostly breast milk of nursing mothers (El-Nezami et al., 1995; Zarba et al., 1992) and has lower toxicity than AFB1. AFM1 can result into DNA damage, gene mutation, cell transformation in mammal cells, insects and animals (Creppy 2002; Govans et al., 2002; Prandini et al., 2009).

AFM1 has been a major concern to health effect due to its frequent occurrence (Zhang et al., 2015). The normal value of AFM1 in Europe is between 0.01mg/kg and 0.05mg/kg.

In an in vitro study, AFB1 and AFM1 causes DNA damage of differentiated caco2- cells (Zhang et al., 2015). Consumption of AFB1 and AFM1 for a longer period of time can impair or causes the risk of injury towards the intestinal tract.

AFLATOXIN G: The aflatoxins G1 and G2 are named after the greenish yellow they fluoresce under UV light.

Other metabolites of aflatoxin include aflatoxicol, aflatoxicol H1, aflatoxins P1 and Q1. AFG21 is found higher in concentrations than AFB2 and AFG2 (Weidenborner, 2001).

2.1.6 BIOCHEMISTRY OF AFLATOXIN

2.1.6.1 BIOSYNTHESIS OF AFLATOXIN

The biosynthesis of aflatoxin is by a typeII polyketide synthase, the first step is the norsolorinic acid, an anthraquinone (Bennette et al., 1997). Sterigmatocystin is final biosynthetic product in the patway.

Fig 2.1.6.1 Biosynthesis of aflatoxin   (Bennette et al., 1997)

2.1.6.2 METABOLISM OF AFLATOXIN

The main metabolic site of aflatoxin is the liver. Phase I and phase II metabolic reactions exist as the major types of aflatoxin metabolism. Oxidative, reductive, or hydrolytic processes occur in phase I reaction and necessary chemical structure are provided for phase II reactions. Phase I may lead to activation and detoxification of a compound and mostly mediated by the cytochrome P450 (CYP450) enzyme systems while phase II reactions may lead to detoxification or formation of biochemical lesions. Phase II metabolism includes sulphate, glutathione, and amino acid conjugation reactions (Monosson, 2012).

 

Phase I aflatoxin metabolism

CYP450 and isoforms of enzymes oxidize AFB1 to several products. Phase I bioactivation pathway for AFB1 is the CYP450-mediated oxidation to reactive AFB1-8,9-epoxide (Swenson et al., 1977). There is reaction between AFB1-8,9-epoxide and N7 atom of guanine to form a pro-mutagenic DNA adduct (AF-n7-guanine). The DNA adducts are of fairly resistance to DNA repair processes, and this can cause gene mutation and thus developing into cancers mostly the hepatocellular carcinoma (Wild et al., 2000).

CYP450 plays a main role in the AFB1 activation, which occurs due to the presence of high level of enzyme.

Fig.2.1.6.2 Metabolism of aflatoxin in the liver (Wild et al., 2000)

PHASE II

Phase II reactions involve conjugation to glucuronic acid, sulphate, and GSH. The phase I metabolism undergo phase II enzymatic metabolism by glutathione-S-transferase (GSTs) that involves in the catalysis of conjugation reactions. Aflatoxin can be conjugated with SH groups after phase I reactions, thus giving rise for further detoxification and elimination of the toxin (Wang et al., 1999).

Fig.2.1.6.3 Aflatoxin B1 (AFB1) metabolism (Wang et al., 1999)

2.1.7 EFFECTS OF AFLATOXIN

The exposure of aflatoxin to hepatocytes in vivo caused swelling, formation of bleb, polymorphic condition and lysis. It was reprted that Aflatoxin B induces cytoxicity on hepatoma cells of mouse. Also the suspension of red blood cells (RBC) treated with aflatoxin in vivo resulted in a concentration-dependent swelling and lysis (Verma and Raval, 1991)

The effect of aflatoxin is to result in the reduction biosynthesis of protein by forming adducts with DNA, RNA, and protein. This allows inhibition of RNA synthesis and DNA dependent RNA polymerase, thus causing a degranulation of the endoplasmic reticulum (Newberne 1994; Groopman et al., 1996)

Aflatoxin can cause enterocyte damage and can contribute to leaky gut. It has been reported to be a potent immunosuppressant in relevant animal model (Mohsenzadeh et al., 2016; Pitt et al., 2012; Raissuddin et al., 1993; Wild et al., 2015).

Afaltoxin can also cause liver toxicity which may damage the production of insulin like growth factor pathway proteins resulting in adverse effect on child growth (Ubagi et al., 2010; Wild et al., 2015).

2.1.7.2 CARCINOGENIC EFFECTS OF AFLATOXIN

Aflatoxiins are major risk of liver cancer; it was identified as a Class 1 carcinogen by the International Cancer Research Institute (Gorelick, 1993). It was also reported that it is the major cause of hepatocellular carcinoma and cholangiocarcinoma (Liu et al., 2008)

The deletions of mutation in the P53 tumor-supressing gene the activation of dominant oncogens are risk of hepatomas (Dragan, 1993). The mechanism of aflatoxin toxicity involves the formation of DNA adduct AFB1-8,9-exo-epoxide(AFBO) (Bedard and Massey, 2006; Hamid et al., 2013) which can result into DNA damages and induces aflatoxicosis when it binds to protein amino acids in vitro (Hamid et al., 2013).

The mutagenic and genotoxic effects of AFB1 is as a result of the following p450 which are; CYP3A4, CYP1A2, CYP3A5 epoxidation of AFB1 to highly reactive exo-8,9-epoxide form that lead to intercalation between DNA base pairs and reacts with the guanyl N7 atom (Wojnowski et al., 2004). Depurination releases an AFB1-N7-guanine adduct that is eliminated in urine. In covalent modification of DNA, AFB1 8,9- epoxides can form covalent adducts with serum albumin  following rapid hydrolysis in blood to AFB1-dihydrodiol in equilibrium with the lysine reactive AFB1-dialdehyde(Johnson et al., 1996)

Fig.2.1.7.2 Mechanism of toxicity of aflatoxin

2.1.8 TOXICOLOGY OF AFLATOXIN

Aflatoxin B1 is the major and the most potent form of the toxin, the toxin is processed through a lot of competing pathways in animals.

Aflatoxicosis is the poisoning that occurs as a result of aflatoxin ingestion. The two major forms of aflatoxicosis are acute and chronic aflatoxicosis. The acute severe intoxication can lead to liver damage, illness or death. The symtoms include; haemorrhagic liver necrosis, bile duct proliferation, edema. In both the chronic and acute aflatoxicosis, the main target organ is the liver (Eaton et al., 1993).

Fig.2.1.8 Aflatoxin pathway in animal metabolism (Eaton et al., 1993)

2.1.9 WAYS OF REDUCING AFLATOXIN EXPOSURE

Public awareness should be made on the toxin, health risks and control methods that might help minimize its prevalence while extending scientific findings to the wider public for massive national development (James et al., 2017; Jolly et al., 2006).

Also, food dealers should be enlightened; communicating to them in a language they can easily understand using appropriate materials that will enhance dissemination of information. Professionals with adequate knowledge should be highly involved (Jolly et al., 2009).

Management practices such as timely planting, drought stress control, weed and pest control, early harvesting, good sanitation and proper cleaning should be adopted in order to eradicate the lowest degree conditions and factors that promote fungal infestation and aflatoxin infection (Wagacha and Muthomi 2008).

The practice of crop rotation should also be adopted to help reduce aflatoxin prevalence in crops by breaking the cycles and builds up of toxin producing crops. Cultivation of resistant varieties would help in the control of the spread of toxins. This can be made possible when aflatoxin inhibitory compounds are identified and used in breeding of new cereal and leguminous varieties (Hell and Mutegi, 2011). Therefore, knowledge of resistant varieties should be sought from plant breeders before cultivation (Commission, 2004).

There should be adequate cereal and legumes dryings after harvest in order to reduce moisture content of about of 10% which may help to limit toxin production (Hell and Mutegi, 2011; Atanda et al., 2011). Well-controlled conditions such as temperature and relative humidity should be properly check for in order to minimize the activity of fungal and the development of aflatoxin in cereals and legumes.

Also, appropriate building storage structures that control temperature and relative humidity in order to ensure effective drying of food crops. Traditional techniques that may include field and bare ground drying contribute to fungal contamination (Okello et al., 2010).

Regulatory bodies or agencies must ensure food commodities on the market contaminated with aflatoxin above the limit that is allowed are being monitored without compromising the set standards (Covic and Hendriks, 2016). Routine inspection of aflatoxin levels in foods on the market and those stored by farmers should be adopted (Covic and Hendriks, 2016).

Researchers should bring about better and efficient ways of how to utilize aflatoxin contaminated food produce; this will ensure that the farmers and the food vendor’s effort are not in vain (Commission, 2004). Contaminated food with aflatoxin can be processed into another product. For example, groundnut should be processed into oil for human consumption.

There should be a reduction of aflatoxin-prone food products such as maize and groundnut products. This may help to control its prevalence in humans. Alternative food crops such as root and tuber food crops should be encouraged (Amagloh and Hardacre, 2012).

Application of ozone for aflatoxin reduction can also be adopted, ozone is a triatomic oxygen formed by the input of a high energy to oxygen. It is a strong oxidant that decontaminates fresh produce, juice and also helps in the degradation of chemical toxicants and pesticides. Ozone can either used as a gas or after dissolving in water (Karaca and Velioglu, 2007). Freitas-Silva and Venancio (2010) have reviewed the application of ozone in myctoxin decontamination; the result showed that ozone can help in aflatoxin degradation of various food products. The mechanism of aflatoxin degradation by ozone starts with an electrophilic attack of C8-C9 double bond on the furan ring which causes the formation of primary ozoznides followed by rearrangement into derivatives of monozonide such as aldehyde, ketones, and organic acids (Jalili, 2016).

Aflatoxin can also be reduced by electrolyzed water: Electrolyzed water (EW) is generated by the electrolysis of dilute NaCl or KCl-MgCl2 solution (Hricova et al., 2008). Suzuki et al., (2002) reported the use of EW to sterilise Aspergillus parasiticus and eliminating mutagenicity of aflatoxin B1 (AFB1) by the OH originating from OHCl.  It was reported there was approximately 90% reduction of AFB1 in peanuts after soaking in neutral EW for 10 minutes and 15 minutes in acidic EW, although soaking in basic EW was rendered ineffective (Xiong et al., 2012).

Fig.2.1.9 The mechanism and degradation of aflatoxin by Electrolysis of water (Escobedo-Gonzalez et al., 2016)

2.2 PROTEIN MALNOURISHMENT

Malnutrition can be termed as an imbalance between the food taken and the ingested amount required by the body in order to ensure the most favourable growth and function (Fock et al., 2010). It has been reported that more than 100 million children worldwide suffer from malnourishment (FAO, 2012).

Several discoveries have been made that severe malnutrition is linked with differential increased levels of DNA damage which depends on each tissue’s structural and functional characteristics (Bentancourt et al., 2005).  Severe malnutrition has been linked with an increase in lipid peroxidation (LPx) in children’s erythrocyte and serum (Bosnak et al., 2010).

Malnutrition has also been linked with decreased DNA repair capacity (Gonzalez et al., 2002). Imbalance in the superoxide radical, alteration in the distribution of neurotransmitters receptors (Morgane et al., 2012) can be traced to malnutrition.

Protein malnutrition is a major form of malnutrition which is described as an imbalance between food intake and that needed by the body to ensure optimal growth and function (Fock et al., 2010; Bossola, 2015). Protein malnutrition has been reported to be a common feature in cancer (Tarek et al., 2013).

Protein malnutrition (PM) can lead to delay in body maturation, neurologic and musculoskeletal system development (Myneris-perxachs et al., 2016). PM can also lead to liver biochemical characteristics and histology (Caballero et al., 2011; Ronchi et al., 2010; Ronchi et al., 2004).

Reports has been made that a poor consumption of amino acid can lead to oxidative stress (Cabarello et al., 2011; Ronchi et al., 2010) whereby the antioxidant properties are overpowered, reactive oxygen species (ROS) injure cells in a way that can result into death by necrosis or apoptosis (England and Cotter, 2005). There was a high increase in oxidative damage to lipids and proteins in an experiment to determine the effect of protein malnutrition on oxidative status in rat (Ana et al., 2006).

2.2.1 EFFECTS ON PROTEIN MALNOURISHMENT

The inadequate intake of protein can result in kwashiorkor, marasmus and marasmic-kwashiorkor.

The term kwashiorkor is a condition that arises as a result of protein malnourishment or inadequate protein intake (Tasneem et al., 2010). Symptoms that arise include oedema, hair loss, fatty liver disease, anaemia, swollen abdomen and so on.

Marasmus can be seen as a chronic that results from a deficiency in protein, fat or carbohydrate. A marasmic individual is severely wasted in skeletal muscles and fat depots, they are underweight, inactive, and head appears disapropotional in size (Scherbaum and Furst, 2000).

Marasimic- kwashiorkor is a condition with both the features of marasmus and kwashiorkor. It occurs when stress is much on a chronically ill, starved patient. The somatic and visceral protein stores are depleted as well body fats stores.  This condition is associated with high incidence of life threatening complications, people with this condition have high risk of infections and poor wound healing (Scherbaum and Furst, 2000; Touyz, 2000).

2.2.2 POSSIBLE EFFECTS OF PROTEIN MALNOURISHMENT

Protein malnourishment is the major cause in the decrease in serum protein and albumin level. The lipoprotein synthesis cannot take place as a result of protein deficiency leading to fat accumulation in liver and thus resulting in fatty liver. In fatty liver, there is a decrease in albumin synthesis and serum protein. Albumin decreases the plasma colloidal osmotic pressure, leading to edema (Justin, 1991).

Fig 2.2 Mechanism on the possible effects of protein malnourishment (Justin, 1991)

2.2.3 .PREVENTION AND TREATMENTS

Protein malnourishment conditions (Kwashiorkor, marasmus and marasmic-kwashiokor) can be prevented or treated by; Correcting water and electrolyte balance, treatment of infections and worm infestations, dietary supply of 3-4g protein and 200cal/kg body weight/day with vitamins and mineral supply. Also parents should be counselled and plan future that include immunization and diet supplements.

2.2.4 OXIDATIVE STRESS AND OXIDATIVE DAMAGE

Oxidative stress can be defined as an imbalance between antioxidants and pro-oxidant which can be as a result of the decrease in endogenous antioxidants, low intake of dietary antioxidants and increased formation of free radicals (Yilmaz et al., 2017; Halliwell and Gutteridge, 2007). It can increase in lipid peroxidation and also increase in adduct formation with RNA, DNA. Effective oxidants that play a role in the protection of tissues from harmful effects of AFB1 are GSH and GST (kobertz et al., 1997)

Oxidative DNA damage is the changes in DNA structures and functions as a result in the interaction of reactive oxygen species (ROS) with DNA. It can be regarded as one of the damages caused by AFB1 and can be induced by reactive oxygen species (ROS) leading to tissue damage via different mechanism such as DNA damage, lipid peroxidation, protein oxidation and thiols depletion (Shen et al., 1994). The formation of C8-OH –adduct radical as a result of the connection of ·OH to C8 and altered to 8-OH-guanine (8-OH-Gua) by one electron oxidation (Kasai et al., 1992). AFB1 has the tendency to convert into the epoxide and DNA adducts that result in the formation of DNA strand breaks and mutations (Valavanidis et al., 2009). CYP450 enzyme system activate AFB1 to form AFB1-8,9-epoxide which connects to nucleophilic sites in DNA to form 8,9-dihydro-8-(N7)-9-hydroxy-AFB1 (AFB1-N7-Gua). 8-OH-Gua glycosylase is mainly the enzyme for the repair of 8-OH-Gua in short-patch base excision repair in humans. 8-OHdG is a major biomarker of oxidative DNA damage (Valavandis et al., 2009)

Fig.2.2.4 Metabolic activation of Aflatoxin B1 (Kobertz et al., 1997)

2.2.5 FREE RACICALS AND LIPID PEROXIDATION

Free radicals are reactive species with an unpaired electron. For example hydroxyl (.OH) and superoxide radicals (O ̄2) can cause tissue damage.   Oxygen derived free radicals are commonly termed as reactive oxygen species (ROS). The mitochondrial electron transport system, peroxisomal fatty acid, CYP450, and phagocytic cells are the primary sites where ROS are produced (Ray et al., 2012). The reaction of DNA with ROS cause breaks in the DNA and mutation, which could result in carcinogenesis.

Free radicals exhibit short half- life while lipid peroxidation has long half-life, thus they easily diffuse and cause oxidative damage away from their site of production (Muller et al., 2007).

Fig 2.2.5 Endogenous sources of reactive oxygen species (ROS) (Hardas, 2012)

2.3 EFFECTS OF AFLATOXIN ON OXIDATIVE STRESS AND DNA DAMAGE

Oxidative stress may be as a result of the aflatoxin or its metabolites, it also play key role in aflatoxicosis. AFB1 is activated by CYP450 to AFB1-8, 9-epoxide in the liver forming adducts with both DNA and protein. It has been reported that CYP450 enzymes generate superoxide hydrogen peroxide which can cause oxidative stress (Shimamoto, 2013; Sun et al., 2013). It is considered to be the main factor in initiating and progressing liver cirrhosis.

The oxidative damage can be caused by aflatoxin considered to be the main mechanism leading hepatoxicity (Preetha et al., 2016). It has been revealed thatAFB1 leads to oxidatve DNA damage in rat liver.

Fig 2.3 Effects of aflatoxin on oxidative stress (Marin and Teranu, 2012)

2.3.1 EFFECTS OF PROTEIN MALNOURISHMENT ON OXIDATIVE DAMAGE

The effects protein malnourishment has on oxidative tissue damage may be as a result of inadequate of protective and repair mechanism in protein deficient animals or humans. Poor consumption of amino acid may cause oxidative stress (Caballero et al., 2011; Ronchi et al., 2010). Oxidation occurs at specific amino acid such as histidine, lysine, proline, tryptophan, methionine, tyrosine and phenylalnine residues (Requena et al., 2001). Oxidized proteins aggregate and form cross-links, thereby leading to the prevention of proteasome action and the accumulation of oxidized proteins (Yin, 1995).

It has also been reported that there is linkage between the increased levels of DNA damage and severe malnutrition which depends on each tissue’s structural and functional characteristics (Bentancourt et al., 2015).

 

 

 

CHAPTER THREE

MATERIALS AND METHODS

3.1 MATERIALS

3.1.1 Chemicals and reagents

Chemicals used in the study of this research work are 1-chloro-2,4-dinitrobenzene (CDNB), thiobarbituric acid (TBA), pyrogallol, glutathione, trichloroacetic acid (TCA), sodium hydroxide, manganese chloride, 5,5’-Dithiobis(2-nitrobenzic acid)(DTNB), Zinc sulphate

Reagents used in this study include ethanol, methanol, 1XTBA, thiol buffer, 1XPBS, Griess, fox, GST buffer, SOD buffer.

3.1.2 Laboratory equipment

EQUIPMENT	MODEL	MANUFACTURER
Spectrophotometer
Waterbath	SBS40	Stuart
Microcentrifuge
Micropipette
Dissecting kit
Disposable sterile syringe kit
Weighing balance
Homogenizer

3.1.3 Reagents, Chemicals and Manufacturer

REAGENT NAME	MANUFACTURER
Ethanol	Sigma
Sodium Chloride	Riedel-de-Haen
5,5’-dithio(2-nitrobenzoic acid	Sigma
Hydrogen peroxide	Sigma
1-chloro-2,4-dinitrobenzene	Sigma
Glutathione	Sigma
Diethyl ether	Sigma
Pyragallol	Sigma
Trichloroacetic Acd	Riedel-de Haen
Hydrochloric Acid	Analar
Ethylenediaminetetracetic acid	Naafco

3.1. Other materials

Wash bottles, Ependoff tubes, Pipette, pipette tips and beaker

3.1.4 Animals

Thirty-two albino rats were used for the experiment and they were obtained from the animal house of the Federal University of Agriculture, Abeokuta, Ogun State. All the rats were in cages at a room temperature of 30oC in the animal laboratory. They were fed with aflatoxin induced diet and protein malnourished diet.

3.1.5 Experimental design

Thirty-two albino rats were used for this study. They were grouped into eight groups

GROUP	GENDER
A	Male	Control
B	Female	Control
C	Male	Aflatoxin diet
D	Female	Aflatoxin diet
E	Male	Protein malnourished diet
F	Female	Protein malnourished diet
G	Male	Protein malnourished +Aflatoxin diet
H	Female	Protein malnourished +Aflatoxin diet

3.2. METHODS

3.2.1. Weight of animals

3.2.2. Sample collection

The blood samples of the rats were collected via cardiac puncture under mild anaesthesia with the use of diethyl ether. Heparin was added into the syringe to avoid blood coagulation. The liver and kidney of the rats were collected, 0.1g of each were weighed and homgenized.

3.3 BIOCHEMICAL ASSAY

3.3.1 DETERMINATION OF LIPID PEROXIDATION

THIOBARBITURIC ACID REACTVE SECIES

Assay condition: Wavelength 535nm, Temperature 100oC

Materials and reagent used: Thiobarbituric acid reagent, water bath or ecotherm and UV spectrophotometer.

Procedure

Lipid peroxidation test was determined using the method of Buege and Aust (1978).

Treat 0.05ml of sample (diluted water for blank) with 0.1 ml of 1X TBA reagent and incubate in boiling water for 15mins. Tube was immediately placed under running tap to cool. Centrifuge at 1000rpm for 10mins and read absorbance of clean supernatant against blank at 535nm.

Calculation

Concentration (M) = Absorbance/E

E is the extinction coefficient

1.56x 10⁵ /M/cm is the extinction coefficient of MDA-TBA complex at 535nm

3.3.2 LIVER FUNCTION TESTS

ALANINE AMINOTRANSFERSE (ALT)

Principle

The determination of ALT activity is according to the stated reactions

Alpha-ketoglutarate +alanine → Glutamate+pyruvate

Pyruvate +NADH + H LDH Lactate +NAD

The rate at which NAD is consumed is determined photochemically and directly proportional to the ALT activity in the sample

Assay condition: Wavelength 340nm, Temperature 25C, 37C

Procedure

                                                BLANK                              SAMPLE

Sample                                       ─                                        0.02ml

Solution R1                                ─                                        0.01ml

Distilled water                           ─                                         ─

The solution would be mixed and incubated for exactly 30minutes at 30oC.

0.01ml of solution R2 would be added, mixed and incubated for exactly 20 minutes at 250C.

In the already incubated mixture, NaOH was added and the absorbance was read after 5 minutes.

DETERMINATION OF ASPARTATE AMINOTRANSFERASE

Assay condition: Wavelength 340nm, Temperature 25C

This test was carried out with the use of AST reagent kit

Principle:The kinetic determination of AST activity is stated below

Alpha ketoglutarate +Aspartate   →  Glutamate + oxaloacetate

Oxaloacetate + NADH     →    MDH  Malate + NAD+

The rate of NADH consumption is determined photometrically and is directly proportional to the AST activity in the sample

DETERMINATION OF ALKLINE PHOSPHATASE

Assay condition: Wavelength 340nm, Temperature 25C

Principle

The hydrolysis of P-nitrophenyl phosphate is catalysed by alkaline phosphatase at pH 10.4, giving p-nitrophenol + phosphatase according to the stated reactions:

P-nitrophenylphosphate +H20   →     P-nitrophenol + phosphate

Calculation

ALP (U/I) = change in absorbance/min x 3300

Procedure

A mixture of 100 microlitre of solution R1 and 20 microlitre of distilled water was taken for blank. For sample reading, 20 microlitre of sample and 100 microlitre of solution R1 was added. Each solution were mixed by inversion and incubate for exactly 30 minutes at 37 degree Celcius. 100 microlitre of solution R2 was added in each tube, they were mixed and incubated for exactly 20 minutes at 25 degree Celcius.

3.3.4 DETERMINATION OF ALBUMIN

Assay Condition: Wavelength is 630nm, Temperature is 37C

Principle

Serum albumin measurement is based on its quantitative binding to the indicator 3’,3’,5’,5’-  tetrabromo- m c BBB    B BB   resolsulphonephthalein (BCG, bromocresol green).

Procedure

Using ependoff tubes, pipette;

Blank                   Standard                  Sample

Distilled water          0.005ml                   –                              –

Standard                     –                             0.005ml                   –

Sample                       –                              –                             0.005ml

Reagent                     1.5ml                       1.5ml                     1.5ml

Incubate for 5 minutes at 37C after mixing. The absorbance of sample (A sample) and standard (A standard) is recorded against the reagent blank.

3.3.5 DETERMINATION OF GLUTATHIONE LEVEL

Assay condition: Wavelength: 412nm, Temperature: 25C

Principle: According to the method of Ellman (1959)

Procedure:

A mixture of 125 microlitre of sample, 100 microlitre of water and 25 microlitre of 50% trichloroacetic acid (TCA) was centrifuged at 5000rpm for 15 minutes. 200 microlitre of the supernatant was taken and mixed with 400 microlitre of thiol buffer. 100 microlitre of DTNB was added and the absorbance reading was read against blank after 5 minutes at 412nm.

3.3.6 DETERMINATION OF CHLOROMINE LEVEL

Assay condition: Wavelength: 412nm, Temperature: 25C

Procedure

0.2g of NaOH was weighed in 100mL of H2O (5mM of NaOH), a freshly prepared of 2.0mg of DTNB was dissolved in 5ml of the already prepared 50mM NaOH. The stock DTNB 40 fold was diluted in 1x PBS. A 50 microlitre of sample and 50 microlitre was added to 1 mL TNB reagent for blank. The mixture of each tube was incubated for 15 minutes in the dark and the absorbance reading was taken at 412nm.

 

 

 

 

3.3.7 DETERMINATION OF NITRIC OXIDE LEVEL

Assay condition: Wavelength: 540nm, Temperature: 25C

Principle: Using a modification of the method by Yucel et al., 2012, nitrite concentration in 10% Red blood cell (RBC) samples was determined by Griess reaction. The measure of the amount of nitrite as an index of the amount of nitric oxide in a sample was as a result of the use of Griess method.

Procedure:

A mixture of 50 microlitre of sample and 75 microlitre of distilled H20 was added to 125 microlitre of 0.3 normal NaOH and leave to incubate at room temperature for 5 minutes. Also, 6.5 microlitre of 10% ZnSo4 to deproteinize was added to the mixture and centrifuged at 4000rpm for 3 minutes. 200 microlitre of the supernatant was added to 200 microlitre of Griess reagent. The absorbance of the mixture was read after 30 minutes incubation.

3.3.8 DETERMINATION OF HYDROGEN PEROXIDE LEVEL

Assay condition: Wavelength: 560nm, Temperature: 25C

Principle:

The FOX method involves the oxidation of ferrous to ferric iron by hydroperoxides under certain acidic conditions at room temperature. Xylenol orange [3,30-Bis (N,N – bis(carboxy-methyl)aminomethyl)-o-cresolsulfonephthalein] tetrasodium salt binds ferric ion to form a chromophore complex which absorbs strongly at 540-600nm. This is illustrated in the equation below:

Fe 2+  + ROOH → Fe 3+  +OH- + RO.

Fe3+  + XO →Fe- XO

Procedure:

A mixture of 50 microlitre of sample and 25 microlitre of fox reagent was added in 450 microlitre of distilled water in eppendof tubes. The total mixture was incubated in the dark at room temperature. The absorbance was read against blank at 560nm.

3.3.9 DETERMINATION OF SUPEROXIDE DISMUTASE (SOD) LEVEL

Assay condition: Wavelength: 420nm, Temperature: 25C

Procedure:

According to Marklund and Marklund (1974), superoxide dismutase was determined.  The mixture of 100 microlitre of buffer, 830 microlitre of distilled water and 50 microlitre of sample was incubated at room temperature for 10 minutes. 20 microlitre of pyrogallol was added to the already incubated and read at 420nm for 3 minutes at 1 minute interval.

Calculation:

Superoxide activity is calculated as;

% inhibition = (∆A 420nm/minute of blank ─ ∆A 420nm/minute of sample) x 100

∆A 420nm/minute of blank

Units of SOD =      % inhibition

(100- % inhibition) 

Units/mL= Units x dilution factor

Units/mg protein= Units/mL

Mg protein/mL

3.3.10 DETERMINATION OF TRIGLYCERIDES LEVEL

Assay condition: Wavelength: 500nm, Temperature: 37C

Procedure:

                                            BLANK                               SAMPLE                      STANDARD

Reagent                               1.0mL                                  1.0mL                                 1.0mL

Sample                                    ─                                      0.005mL                                ─

Standard reagent                     ─                                         ─                                     0.005mL

3.3.11 DETERMINATION OF ALBUMIN LEVEL

Assay condition: Wavelength: 630nm, Temperature: 25C

Procedure:

A mixture of 5 microlitre of distilled water and 1500 microlitre reagent was used for blank. 5 microlitre and 1500 microlitre of reagent was used as standard in another tube. Also, a solution of 1500 microlitre of reagent and 5 microlitre sample was prepared in a separate tube. Each of the tubes were incubated for 5 mins at 25 degrees celcius at 630nm.

3.3.12 DETERMINATION OF TOTAL PROTEIN LEVEL

Assay condition: Wavelength: 660nm, Temperature: 25C

Procedure: According to the method of Lowry (1951). 195 microlitre of distilled water + 1mL of lowry reagent + 5 mirolitre of sample pipetted into an ependoff tube, the mixture was mixed by inversion and incubated for 10minutes at room temperature. Immediately after the incubation, 100 micolitre of Fiolin- ciocalteu reagent was added and incubated for 30 minutes at room temperature. Thereafter, the absorbance was read at 660nm and concentration of protein in mg/dL was calculated with the use of standard curve.

3.3.13 DETERMINATION OF CHOLESTEROL LEVEL

Assay condition:

Procedure:

3.3.14 DETERMINATION OF UREA LEVEL

Assay condition: Wavelength: 546nm, Temperature: 25C, 37C

Procedure:

20 microlitre R1 + 2 microlitre of sample/callibrtaor/distilled water for sample, standard and blank respectively. Mix and incubate for 10 minutes at room temperature. 500 microlitre of R2 and R3 into the mixture, incubate again for 15 minutes at 37C. Read the absorbance.

 

3.3.15 DETERMINATION OF PHOSPHOLIPIDS LEVEL

Assay condition: Wavelength: 600nm, Temperature: 25C

Procedure:

8 microlitre of sample (distilled water for blank) + 800 microlitre of R1, mix and incubate for 5 minutes at room temperature. Read first absorbance. Add 200 microlitre of R2 to the mixture, mix and incubate for 5 minutes at room temperature. Read the second absorbance at the same wavelength.