CHAPTER IV

DISCUSSION

In the present in vitro study, it was found that addition of DEP significantly

increases lipid peroxidation in mice liver and kidney homogenates. The increase was

dose-dependent and almost similar in both liver and kidney homogenates as compared

to controls (Table 3. 1; Figure 3.1 & 3.2). Lipid peroxidation is a complex process

known to occur in animals. It involves the formation and propagation of lipid radicals,

the uptake of oxygen, a rearrangement of the double bonds is unsaturated lipids and

the eventual distraction of membrane lipids, with the production of a variety of

breakdown products (Dianzani and Barrera, 2008). Lipid peroxidation has been taken

as an indicator of cellular oxidative damage (Halliwell et al., 1999). The higher level

of TBARS after DEP treatment was also reported in Wistar rat and olive flounder

(Paralichthys olivaceus), a marine culture fish by Pereira et al. (2006) and Kang et al.

(2010) respectively.

The protein content was significantly decreased in DEP - treated liver and

kidney homogenates. The effect was dose-dependent (Table 3.2; Figure 3.3 & 3.4).

This could be due to oxidative stress. Lipid peroxidation – derived free radical could

attack back - bone of protein and side - chains of specific amino acid residues (Wu et

al., 2013) and might be responsible for significant reduction in protein content in DEP

- treated liver and kidney homogenates.

The result of the present study revealed that DEP treatment for 30 days caused

significant, dose-dependent reduction in body weight of mice (Table 3.4; Figure 3.5).

The decrease in body weight is because of the reduced feed intake and anorexia which

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was seen throughout the treatment period with LD, MD and HD of DEP. Brown et al.

(1978) and Lamb et al. (1987) have reported significant (>10%) decrease in body

weight and body weight gain in experimental animals after acute and intermediate –

duration dietary exposure of DEP. Lamb et al. (1987) reported that dietary

administration of DEP equivalent of 3250 mg/kg b.w./day was associated with 47%

weight gain inhibition. A study by Brown et al. (1978) had explained that the

reduction in body weight could be due to the lower food consumption and/or poorer

food utilization.

Oral administration of DEP for 30 days in the present study caused increase in

absolute as well as relative weights of liver and kidney of mice (Table 3.4; Figure 3.6

& 3.7). A significant increase (12%) in relative liver weight was observed in wistar

rats, reported by Oishi and Hiraga et al. (1980). Brown et al. (1978) reported that

administration of DEP had increased relative liver and kidney weights in both sexes

of mice. This also agreed with Olsen et al. (1982). An increase in relative kidney

weight was observed in male and female rats administered 5% diethyl phthalate in the

diet (Brown et al., 1978). The marked increase seen in liver weight could be due to

increased lipid and cholesterol content (Table 3.5), which has also been proved by

histopathological investigations (Plate E) in the present study revealing fatty

degenerative change (Plate C, D, E, H, I and J) in DEP - treated groups (Group 3, 4

and 5).

Treatment of all three doses of DEP caused significant increase in glycogen

content in liver (Table 3.5; Figure 3.9). Liver is the main site for synthesis and storage

of glycogen. Being a key organ involved in this metabolic process, increase in the

glycogen level would indicate suppressed carbohydrate metabolism. Reduced

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glycogen phosphorylase activity has been observed in liver of DEP - treated mice

(Table 3.7). Accumulations of excessive glycogen due to DEP treatment could lead to

hypertrophy of the hepatocytes, which could lead to complete impairment. Studies by

Sonde et al. (2000) and Pereira et al. (2006) have also reported significant increase in

liver glycogen content.

Diethyl phthalate administration caused significant increase in total lipid

contents in liver and kidney of mice (Table 3.5; Figure 3.11). It might be due to

inhibitory effect of DEP on acetyl-CoA carboxylase enzyme which is required to

catalyse the rate-limiting step in fatty acid synthesis. The enzyme inhibition might be

either direct by an alteration of the enzymes’ structure as a result of interaction

between toxin and enzyme or indirect which may be through a toxin – induced

changes in the cellular levels of allosteric effectors of acetyl-CoA carboxylase

(Beynen and Geelen, 1982). PPAR-α (Peroxisome proliferator activated receptor

alpha) is a transcription factor and a major regulator of lipid metabolism in liver and

kidney, probably DEP exposure results in significant decrease in the β-oxidation

process hence accumulation of fat or lipid occur in the major metabolic organ like

liver and kidney (Mapuskar et al., 2006).

Table 3.5 shows increased level of cholesterol content in liver and kidney of

mice. Synthesis and metabolism of cholesterol takes place mainly in liver following

which it is transported to other organs. Highly significant increase in cholesterol level

in liver and kidney indicates that DEP causes impairment in cholesterol metabolism.

Some recent reviews on the role of StAR protein (Steroidogenic acute regulatory

protein) of mitochondrial outer membrane indicate it as a regulator of cholesterol

metabolism as well as steroidogenesis and cytochrome P450 side - chain cleavage

79
enzyme (P450scc), which is located on the matrix side of the inner mitochondrial

membrane involved in transport of cholesterol and its metabolites for steroidogenesis

(Nakae et al., 1997; Stocco et al., 2001). As per this we assume that DEP affects the

StAR protein of mitochondrial outer membrane, hence accumulation of excessive

cholesterol is observed. This results correlates well with earlier study in which male

Sprague-Dawley rats administered with 50 ppm DEP through water shows in

significant increase in liver cholesterol content (Sonde et al., 2000). Many other

investigators (Mapuskar, 2007, Pereira, 2006) have reported increase cholesterol

content in liver. Histopathological studies also revealed fatty accumulation in liver of

DEP treated mice (Plates C, D and E).

Thirty days treatment of DEP in mice had resulted in significant, dose-

dependent reductions in DNA, RNA and protein contents in the liver and kidney of

mice (Table 3.5; Figure 3.12 & 3.13). DEP may affects biomarkers by forming DNA

adducts (NAS/NRC 1989). NTP (1993) Board draft showed that DEP produced

concentration- dependent increase in the number of relative sister chromatid exchange

per chromosome in the presence of S9 fractions from rat liver homogenates. The

positive sister chromatid exchange test might indicate a potential for DNA damage.

Phthalates and /or their metabolites have been reported to damage DNA as assessed

by the alkaline comet assay. Two recent epidemiologic reports involving men

attending an infertility clinic showed that sperm DNA damage by DEP metabolite

(Duty et al 2003; Hauser et al 2007). DEP is known to produce oxidative stress in the

biological systems (Sun et al., 2012). Present in vivo and in vitro studies also reports

significantly increased lipid peroxidation (Table 3.1, 3.2, 3.8 and 3.13). That could be

the reason for the reduction in DNA, RNA and protein contents. Nucleic acids i.e.

RNA and DNA are the major group of ROS targets. Oxidative DNA damage includes

80
modification of bases, DNA strand breaks and interstand crosslink (Miwa et al 2008).

DEP generated free radicals causes RNA damage could be due to incorporation of

oxidized nucleotides during the process of RNA synthesis. DNA microarray analysis,

followed by qrtPCR corroboration of the microarray data, demonstrated here that

exposure of human cells to DEP in vitro is consistent with initiation of decreased

expression of a large number of genes that have been identified as essential for fetal

brain development (Hokanson et al., 2009). Reduction in nucleic acids also affects

process of transcription and translation, ultimately causing reduction in tissue protein

synthesis. Proteins are cellular targets of ROS (Starke-Reed and Oliver, 1989; Barnes

et al., 2008). Oxidative modifications such as breakdown of peptide bonds, can

damage protein structures (Stadtman, 2001) that related to the reduction of protein by

elevated oxidative free radicals.

Oral administration of DEP to mice for 30 days had significantly altered the

energy status. DEP treatment resulted in reduction in SDH activity – an enzyme

bound to inner mitochondrial membrane, which could be due to structural and

functional disorganization of the mitochondrial assembly (Fig .3.15). Srivastava et al.

(1978 and 1977) reported that di (2-ethyl hexyl) phthalate (DEHP) also found to

inhibit the activity of total and Mg+ - stimulated ATPase activity in rat liver. Beside

liver, the activity of SDH and ATPse was also inhibited in rat heart, kidney

(Srivastava et al., 1977), lung and gonads (Seth et al., 1976), indicating that

suppression of energy- linked reactions may be a generalized effect of DEHP.

Alteration in mitochondrial potential decreases the rate of cellular ATP synthesis and,

thus nucleotide synthesis which may cause the reduction in DNA and RNA contents.

Energy deficiency of the cell characterised by reduced activity of SDH and ATPase

could be well correlated with reduction in protein content (Panet and Altan, 1979).

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Mitochondria contains biochemical machinery for oxidation of various biomolecules

and produced energy is captured in the form of ATP. Phthalates inhibited the

respiration of isolated mitochondria from rat liver primarily by uncoupling oxidative

phosphorylation (Inouye et al., 1978; Melnick et al., 1982). Other researchers have

suggested that the phthalates inhibited electron transport or energy transport (Ohyama

et al., 1976). Dibutyl phthalate and dimethyl phthalate inhibited the activities of SDH

and ATPase, enzymes of the rat liver inner mitochondrial membrane (Srivastava et

al., 1977; Tanaka et al., 1978; Melnick et al., 1982).

Diethyl phthalate treatment had significantly altered activities of liver marker

enzymes (Table 3.16) indicative of hepatocellular membrane damage and necrosis.

The present study revealed that ALT and AST activities significantly increased in

DEP - treated animals as compared to controls. These two enzymes are localized

normally within the cells of the liver, heart, kidney, gill, muscle and other organs

(Wells et al., 1986). These enzymes are important markers in assessing and

monitoring liver damage (Drotman et al., 1978). AST is a chief mitochondrial

enzyme; damage to the mitochondria would result in leakage of this enzyme into the

serum causing elevation in the serum enzyme levels, which were evident in all the

treated groups. According to Gao et al. (2004), ALT activity is an important index to

measure the degree of cell membrane damage, while AST is an indicator of

mitochondrial damage since it contains 80% of this enzyme. Ghorpade et al. (2000)

reported increased ALT and AST activities in liver and muscle of freshwater fish

Cirrhina mrigala. Mapuskar et al. (2007) and Barse et al. (2007) also reported level of

ALT and AST increased in mice, cyprinus carpio respectively. These are of major

importance in assessing and monitoring functional status of the liver.

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 Alkaline phosphatase (ALP) activity significantly and dose - dependently

increased in serum of DEP - treated animals as compared to control and vehicle

control. Alkaline phosphatase is a marker enzyme for plasma and endoplasmic

reticulum (Wright et al., 1974; Shahjahan et al., 2004) is often employed to assess the

integrity of plasma membrane (Akanji et al., 1993) Pereria el al. (2006). Ghorpade et

al. (2000) and Barse et al. (2007) also reported increase ALP activity. Lipophilic DEP

interact with plasma membrane and thereby could have led to increase in ALP activity

(Barse et al., 2007).

Acid phosphatase (ACP) activity in its own case is a marker enzyme for the

lysosomal integrity (Collins et al., 1971). The present study revealed significant, dose

– dependent increase in ACP activity in serum of DEP - treated mice (Table 3.6).

Similar changes were also reported by Mapuskar et al. (2007) in Swiss mice and

Gorpade et al. (2000) in freshwater fish Cirrhina mrigala. It might be due to the

consequence of damage to lysosomal integrity in the liver. The significant rise seen in

the ACP activity after toxin administration may be attributed to an increase in cellular

degeneration and other pathological liver injury (Verma et al., 2004).

Oxidative stress reflects an imbalance between the systemic manifestation

of reactive oxygen species and a biological system's ability to readily detoxify the

reactive intermediates or to repair the resulting damage. Disturbances in the

normal redox state of cells can cause toxic effects through the production

of peroxides and free radicals that damage all components of the cell,

including proteins, lipids, and DNA. Further, some reactive oxidative species act as

cellular messengers in redox signaling. Thus, oxidative stress can cause disruptions in

normal mechanisms of cellular signaling. Oxidative stress is a condition in which the

generation of reactive oxygen species (ROS), a ubiquitous by - products of aerobic

83
metabolism, overwhelm the cellular antioxidant defence mechanism. ROS have a

crucial role in human physiological and pathophysiological process (D’ Autreaux et

al., 2007).

Most xenobiotics damage liver and kidney by inducing, directly or indirectly

lipid peroxidation. Lipid peroxidation is a major harmful consequences of reactive

oxygen species (ROS) formation (Ambrosio et. al., 1991; Lucas et al., 1998). Results

shown in Table 3.8 & 3.13 indicate significant and dose - dependent increase in lipid

peroxidation in liver and kidney of DEP - treated mice as compared to vehicle control.

It could be due to either increased production of ROS by DEP and/or suppression of

antioxidative defence system resulting in altered redox potential of cell causing lipid

peroxidation and therefore, suggesting a considerable oxidative stress. Pereira et al.

(2006) have correlated increased lipid peroxidation levels with the increased number

of peroxisomes which indicates high level of ROS production due to continuous liver

and kidney insult by DEP.

Increased lipid peroxidation could also be due to impairment in enzymatic and

non-enzymatic antioxidant system in the tissues. Total glutathione (GSH) and total

ascorbic acid (TAA) contents are important endogenous free radical scavenger and

non-enzymatic antioxidants. Oral administration of DEP for 30 days has resulted

significant reduction in level of GSH and TAA contents (Table 3.8 & 3.13).

Glutathione functions as an electrophile, radical scavenger and a redox partener

(Meister et al .1985). This reduction would result in increased free radical injury in

tissue leading to extensive tissue damage. Pereira et al. (2006) had shown reduction

in GSH content in DEP exposed wistar rats which support our study. During free

radical scavenging action, ascorbic acid, transformed into L-dehydroascorbate back to

84
ascorbate (breimer, 1990). The fall in the level of reduced glutathione decreases

conversion of L-dehydroascorbate and probably explains the lowered level of TAA in

DEP- treated animals.

Activities of enzymatic antioxidants (superoxide dismutase, catalase,

glutathione peroxidase, glutathione reductase and glutathione transferase) were found

to reduce with DEP treatment for 30 days (Table 3.9; Figure 3.20 and 21), which

could be due to increased production of free radicals characterized by increased MDA

content. These enzymes are known to scavenge free radicals such as superoxide,

hydroxyl and hydrogen peroxide, thus preventing damage caused by oxidative stress

to the tissue. Superoxide radicals have been reported in several pathological

disorders. A reduction in SOD activity contributes to increasing the level of

superoxide radicals, thus leading to increased oxidative stress which enhances early

cell death. Pereira et al. (2006) also reported depletion of GSH and GR in female

Wistar rats. Diethyl phthalate failed to up - regulate expression of mRNA of the

enzymes SOD, CAT, GST which are induced as a counter measures to the adverse

effects of oxidative stress. These data corroborate recent reports in the literature

(Rosado-Berrios et al., 2011; Erkekoglu et al. 2011, 2012) and suggest that oxidative

stress is a critical mechanism of toxicity for DEP.

Dietary antioxidant intake may be an important strategy for inhibiting or

delaying the oxidation of susceptible cellular substrates, and is thus relevant to disease

prevention in many paradigms. Plants, including herbs and spices, have many

phytochemicals which are potential sources of natural antioxidants. Phenolic

compounds such as flavonoids, phenolic acids and tannins have received attention for

their high antioxidative activity (Rice – Evans et al., 1996). In the present study

qualitative assessment of phytochemicals of hydro-alcoholic seed extract of Nigella

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sativa showed presence of phenols, flavonoids and tannins. Phenolic acids,

flavonoids and tannins are the most commonly found polyphenolic compounds in

plant extracts (Wolfe et al., 2003; Naik et al., 2006). The antioxidative property of

polyphenols is a predominant feature of their radical-scavenging capacity (Facino et

al., 1990; Yang et al., 2001; Cotelle et al., 2001). In the present study quantitative

estimation of crude polyphenols from hydro-alcoholic seed extract of Nigella sativa

revealed presence of significantly high amount of phytochemicals principally

responsible for its protective effect (Table 3.16). Flavonoids are 15- carbon

compounds generally distributed throughout the plant kingdom (Harborne 1988).

Flavonoids and many other phenolic compounds of plant origin have been reported as

scavengers of ROS and viewed as promising therapeutic drugs for free radical

pathologies (Prashad et al., 1998; Chang et al., 2007). Tannins are the most abundant

antioxidants in the human diet and they exhibit many biologically important functions

which include protection against oxidative stress (Atanassova and Christova-

Bagdassarian, 2009). The oxidation inhibiting activities of tannins have been known

for long time (Edeoga et al., 2006).

Hydro-alcoholic Nigella sativa seed extract was found to be potent scavenger

of superoxide, hydroxyl, nitrous oxide and DPPH radicals (Figure 3.41, 3.42, 3.43 and

3.44). Free radical scavenging effect of Nigella sativa had also been reported by

Meziti et al., (2012). These scavenging properties are generally due to high reducing

capacity of polyphenols acting as primary antioxidants (Javanovic et al., 1999).

Nigella sativa is powerful scavenger of DPPH and hydroxyl radical (Burits et al.,

2000). Meziti et al. (2012) reported significant linear correlation between their

superoxide anion scavenging effect and their phenolics and flavonoid contents. Table

86
3.16 showed presence of correlation between total phenolic content and antioxidant

effect of hydro-alcoholic extract of Nigella sativa seed.

Nigella sativa seed extract alone did not have any significant effect on

morphological changes, change in body weight as well as absolute and relative

weights of liver and kidney as compared to untreated and vehicle control. Table 3.19

shows the mitigatory effect of two different doses of Nigella sativa seed extract on

DEP induced body weight reduction. Maximum toxicity had been exerted in high

dose of DEP; cotreatment of Nigella sativa seed extract along with high of DEP

resulted in significant amelioration in body weight of animals. This protective effect

might be due to reduced oxidative stress levels in Nigella sativa seed extract treated

animals resulting in normalization of food intake and metabolism. Kaleem et al

(2006) have shown protective effect of Nigella sativa seed extract on body weight in

diabetic rats. The result indicated reduction in animal body weight under oxidative

stress condition which was successfully ameliorated with cotreatment of two different

dosage of Nigella sativa seed extract (Kaleem et al., 2006).

Oral administration of DEP had significantly increased the absolute and

relative weight of mice liver and kidney. Table 3.20 shows that oral administration of

hydro alcoholic extract of Nigella sativa seed in two different doses significantly

combated these DEP induced changes; maximum effect was observed with high dose

of Nigella sativa seed extract. This protective effect might be due to lipid lowering

effect of the hydro – alcoholic extract of the Nigella sativa seed. Histopathological

studies revealed reduced fatty infiltration in DEP plus Nigella sativa seed extract

treated liver and kidney of mice (Plate M and P). Supplementation of Nigella sativa

seed extract resulted in lowering of the total lipid, cholesterol, triglycerides in the

87
albino rats (Badary et al, 2000; El Dakha khani et al. 2000; Le et al., 2004; Bhatti et

al., 2009).

Diethyl phthalate treatment for 30 days had significantly increased lipid and

cholesterol contents in liver and kidney of mice (Table 3.21). Combined treatment of

Nigella sativa seed extract and DEP caused mitigation in DEP caused alteration in

lipid and cholesterol contents in liver and kidney of mice. The lipid lowering effect of

Nigella sativa seed extract might be due to its phytochemicals which are present in it.

Nigella sativa seed had favourably modified serum lipid proile in rats with significant

decrease in total cholesterol, LDL- cholesterol and triglycerides (Al-L-Ogmani et al.,

2011). Kocyigit et al. (2009) reported that Nigella sativa seed to diet is a remedy that

may prove to be useful in the prevention and treatment of hyperlipidemia and

hypercholesterolemia. This protective effect could be due to presence of some active

ingredients like, saponins which helps in improving serum lipid profile and tissue

lipid and cholesterol contents.

Nigella sativa seed extract alone treatment did not show any significant effect

on protein and glycogen contents of mice. Oral administration of Nigella sativa seed

extract with high dose of DEP caused significant mitigation in DEP caused alteration

in protein and glycogen contents. Alsaif et al. (2007) had reported that Nigella sativa

treatment in ethanol- induced rat increase the total protein level which is in

accordance with our study. Nigella sativa seed decrease the glycogen content in 12

week treated rats (Zaoui et al., 2002).

Diethyl phthalate treatment for 30 days caused significant reduction in nucleic

acid and protein content in liver and kidney of mice which might be due to oxidative

damage. Oxidative DNA damage might be recovered by the antioxidants which can

either stimulate the act of repair enzyme or directly protect against oxidation

88
(Tomasetti et al., 2001). Nigella sativa seed extract is excellent antioxidants and thus

prevents the effect of oxidative stress. The present study shows that cotreatment of

Nigella sativa seed extract along with high dose of DEP significantly mitigated these

changes majorly due to its antioxidative potency. Nigella sativa decreased the DNA

breakage, which may be attributed to its ROS scavenging activity or upregulation of

DNA repair genes which protects against radiation – induced damage by bringing

error free repair of DNA damage (Pereira et al., 2012). Parveen et al., (2011) reported

that Nigella sativa reduced the number of gaps, breaks, exchange, dicentrics and

rings in the chromatid type aberration and chromosome type aberration in bone

marrow cells of rats. Nigella sativa seed contain proteins, alkaloids, saponin and has

been found to increase the level of total protein through stimulation of protein

synthesis (Salem et al., 2005). Besides, Nigella sativa causes suppression of

gluconeogenesis and prevents catabolism of protein and conversion to glucose (Al-

Gaby et al., 1998), and this may lead to increase level of total serum protein

concentration. Oxidative RNA damage is also a feature in xenobiotic –induced

toxicities suggesting that RNA oxidation may actively contribute to the onset or to the

development of disease (Nunomura et al., 2006). DNA and RNA are main

constituents for protein synthesis. Therefore, elevation of protein content was just

because of restored levels of DNA and RNA content by Nigella sativa seed extract

which normalises the process of transcription and translation.

Hepatotoxicity of DEP was evaluated by estimating the activities of various

liver marker enzymes in tissue and serum. As indicated in Table 3.6 activities of ALT,

AST, ALP and ACP were found to increase with DEP treatment in liver tissue as well

as serum of mice. The present result is in agreement with Daba and Abdel Rahman

(1998) who showed, Nigella sativa protective effect against tertiary-butyl

89
hydroperoxide induced hepatotoxicity in rats isolated hepatocytes. Our findings reveal

that administrations of Nigella sativa significantly restores back the levels of liver

marker enzymes (Table 3.22). Nigella sativa treatment protected mice against

hepatotoxicity induced by CCL4 (Badary et al., 1997). Nigella sativa seeds restored

the enzymes level in carbon tetrachloride –induced hepatotoxicity in rats (Iihan et al.,

2005). Gani et al. (2013) showed Nigella sativa extract being able to protect liver

tissues and decrease the leakage of the enzymes (ALT, AST, ALP and ACP) into the

circulations.

All three doses of DEP were found to reduce the activities of SDH and

ATPase activities in liver and kidney of the animals resulting in altered status.

Treatment with Nigella sativa seed extract along with DEP significantly ameliorates

DEP caused changes in the activities of SDH and ATPase in liver and kidney of mice

(Table 3.23). Erşahin et al. (2011) reported that Nigella sativa with its potent free

radical scavenging properties, inhibited subarachnoid-haemorrhage-(SAH-) induced

lipid peroxidation in the brain tissue of rat against the reactive hydroxyl, peroxyl, and

superoxide radicals. In addition, the level of antioxidant glutathione (GSH) was

preserved, thereby ameliorating oxidative damage. The SAH-induced reduction of

Na+/K+-ATPase activity indicated the presence of membrane damage. The Na+/K+-

ATPase is involved in the generation of the membrane potential through the active

transport of sodium and potassium ions in cellular membrane. It maintains neuronal

excitability and controls cellular volume in the central nervous system. Treatment

with Nigella sativa was able to restore Na+/K+-ATPase activity back to normal levels.

Hamed et al. (2013) reported that treatment with black seed alleviated the elevation of

SDH and Na+/k+ ATPase. The restoration of ATPase activity suggest the ability of

Nigella sativa to protect the sulphyhydryl group from oxidative damage through

90
inhibition of lipid peroxidation. Normalised metabolism of protein, carbohydrates and

lipid as well as free radical scavenging effect of plant improves integrity and oxidative

phosphorylation in mitochondria which was highly disturbed in case of energy

deficient state-induced by DEP.

Diethyl phthalate treatment in mice elevated levels of lipid peroxidation in

liver and kidney which was highest with the administration of high dose of DEP

(Table 3.8 & 3.13). Cotreatment of Nigella saiva seed extract along with high dose of

DEP cause significantly reduced levels of LPO as compared to DEP alone treated

animals which might be due to antioxidative and free radical scavenging activity of

Nigella sativa polyphenols as it was well correlated in our in-vitro studies (Table

3.16). The reduced lipid peroxidation level in liver and kidney of Nigella sativa plus

DEP – treated mice could be due to significant elevation in enzymatic and non-

enzymatic antioxidants as well as antioxidative properties of Nigella sativa seed

extract itself. Nigella sativa decreases the lipid peroxidation and increases the

antioxidant defence system activity in the CCL4 treated mice (Kanter et al. 2003).

Nigella sativa seed extract increases GSH and TAA contents in DEP intoxicated

animals (Table 3.24 & 3.29) which could be due to antioxidative properties.

Administration of Nigella sativa restored the activities of nonenzymatic (GSH) and

enzymatic (SOD, CAT, GPx, and glutathione-S-transferase GST) antioxidants as well

as reduced the levels of malondialdehyde (MDA) in the rat brain to normal levels

(Sheikh et al., 2012). Kaleem et al. (2006) reported that oral administration of ethanol

extract of Nigella sativa seeds (300 mg/kg body weight/day) to streptozotocin -

induced diabetic rats for 30 days significantly reduced the elevated lipid and improved

altered levels of lipid peroxidation products (TBARS and hydroperoxidase) and

antioxidant enzymes like catalase, superoxide dismutase, reduced glutathione and

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glutathione peroxidase in liver and kidney. Hydro-alcoholic extract of Nigella sativa

seed is an excellent scavenger of superoxide, hydroxyl, nitrous oxide and DPPH

radical as indicated in our in-vitro studies. This could be the reason for Nigella sativa

seed extract– induced increase in enzymatic antioxidants.

Nigella sativa seed extract -treatment did not show any significant effect on

histopathological changes in liver and kidney of mice as compared to untreated and

vehicle control. Histopathological studies revealed increased in vacuolization, fatty

infiltration and hepatocellular necrosis in the liver of DEP – treated mice (Plate E). In

kidney the histopathological studies revealed degenerative changes, increase

vacuolization, disorganization of glomerulus and increased space between the

glomerulus and the capcule wall in DEP-treated mice (Plate J). This might be due to

the significant reduction in enzymatic and non-enzymatic antioxidants and increased

malondialdehyde (MDA) induced oxidative damage to liver and kidney. Ikele et al.

(2011) also reported histopathological changes in liver and kidney of DEP-treated

Clarias gariepinus. However, co-treatment of Nigella sativa seed extract along with

DEP caused alleviation in diethyl phthalate caused alteration in liver (Plate M) and

kidney (Plate P) of mice. This might be due to antioxidative properties of Nigella

sativa seed extract as results in Table 3.16 shows that conjugate treatment of Nigella

sativa seed extract and DEP decreases lipid peroxidation and increases activities of

antioxidant enzymes. Nigella sativa seeds showed no significant changes in the

hepatocytes of rats (Al- Okbi et al., 1997) and in liver and kidney functions (Ali and

Blundes, 2003). The preventive activity of Nigella sativa may related to its

antioxidants efficiency that inhibit lipid peroxidation (Farrag et al., 2007). Nigella

sativa showed protective effect against lead acetate – induced hepatic tissue damage

in mice (Alarifiet et al., 2012). Aziz Dollah et al. (2012) reported supplementation of

92
Nigella sativa seed to the diets of rats for five weeks did not change the biochemical

parameters of kidney functions as well as histopathological investigation which

illustrated normal architecture of kidney (Le et al., 2004).

Increased hepatocellular damage as observed in the histopathology could be

due to oxidative damage leading to increased free radicals which well correlates with

the increased liver marker enzymes in serum like, ALT, AST, ALP and ACP. It is

also reported by Pereia et al. (2006) in serum DEP-treated rats. Elevated levels of

serum enzymes are indicators of cellular leakage and loss of functional integrity of the

cell membrane in liver (Orisakwe et al., 2003). Thus, their increased presence in

serum may give information on organ dysfunction (Wada et al., 1962). There is no

significant changes in Nigella sativa alone treated animals. Cotreatement of Nigella

sativa seed extract along with high dose of DEP decrease the activities of ALT, AST,

ALP and ACP levels (Table 3.31). Nehar et al. (2012) reported that Nigella sativa

alleviated the increased level of ALT, AST, ALP to the near normal which could be

manifested to reduction in cell membrane disturbance.

Diethyl phthalate also decreased the protein as well as cholesterol contents in

the serum (Table 3.15). No significant changes were observed in all the control groups

as well as Nigella sativa seed extract alone treatment. Impaired cholesterol transport

was evident in the higher dose treated groups due to reduced levels in the serum

cholesterol level. The serum concentration of creatinine is relatively constant under

normal circumstances, unless glomerular filtration rate (GSR) changes, as a result of

defective renal function. Serum creatinine was examined as indicator for kidney

function. Oral administration of DEP for 30 days caused elevation in serum creatinine

content. The increased level of serum creatinine after DEP intoxication (Table 3.31)

might be due to reduced ability of the kidney to eliminate the toxic metabolic

93
substances (Kummer et al., 1988). However, cotreatment of Nigella sativa along with

high dose of DEP leads to prevents the elevation to serum creatinine in kidney of

mice. Al- Okbi et al. (1997) reported that oral administration of Nigella sativa seeds

and extracts caused significant reduction in serum creatinine content.

94