AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: 2151-7517, ISSN Online: 2151-7525, doi:10.5251/abjna.2015.6.2.52.62

© 2015, ScienceHuβ, http://www.scihub.org/ABJNA

Manganese induced Histochemical Localization of Oxygen Derived Free Radicals and hepatotoxicity in poultry birds

B.K. Roy1*, Suruchi Kumari2, and K.K. Singh3

1Dept. of Pharmacology & Toxicology, College of Veterinary Science & A.H., Birsa Agricultural University,

Ranchi-834006, Jharkhand, India, Phone/Fax: +91-651-2450759, E-mail: roybk2001@yahoo.co.in

2Dept. of Pharmacology & Toxicology, College of Veterinary Science & A. H., Ranchi -834006, Jharkhand,

India, E-mail: kdr.suruchi@yahoo.co.in

3Dept. of Pathology, College of Veterinary Science & A.H., Birsa Agricultural University, Ranchi,

Jharkhand, India, E-mail: singhkkpatho@gmail.com

ABSTRACT

Histochemical and histopathological study of liver was conducted to find out the exact mechanism of manganese(Mn

++ ) induced toxicity in poultry birds. Poultry birds were grouped in to three

groups having six birds in each group. Group I was kept as control, group II and group III birds were given Mn

++ @ 50 and 100 mg/kg b.w. orally once daily for 28 days. The exact site of

damage and mechanism of action of Mn++

toxicity was ascertained. The results showed decrease of total protein, indicative liver damage.The liver tissues accumulated Mn

++ in dose dependent

manner. Histopathologial study of liver showed apoptic and necrotic changes characterized by cytoplasmolysis, cytoplasmic eosinophillia, pyknotic karyorrhactic and karyolised nuclei. The above changes were more sever in birds of group III. Histochemical study showed presence of amorphous Mn

++granules in cytoplasm of hepatocytes as blue to black granules. Histochemical

localization of ODFR exhibited very fine formazan granules in liver which were smaller and lesser than Mn

++ bound granules suggestive of not all Mn

++ molecules were active in generation of

ODFR rather some of them might be inactivated after binding with proteins appearing as large granules. It was identified that Mn

++ was primarily accumulated in mitochondria and was

sequesterized there by an energy dependent process. Entry of Mn++

in cells above level of sequesterizing capacity may cause disruption of oxidative phosphorylation and damage due to generation of ODFR exhibiting changes of reversible cellular injury in light microscope.

Keywords: Sub-acute, Manganese, Poultry, Toxicity, Exploration, Mechanism,oxygen derived free radicals.

INTRODUCTION

Manganese (Mn++

) is a mineral element that is both nutritionally essential and potentially toxic. Manganese plays an important role in a number of physiologic processes as a constituent of some enzymes and an activator of other enzymes. Manganese superoxide dismutase (MnSOD) is the principal antioxidant enzyme of mitochondria. Because mitochondria consume over 90% of the oxygen used by cells, they are especially vulnerable to oxidative stress and hepatocytes are rich in mitochondria. The superoxide radical is one of the reactive oxygen species produced in mitochondria

during ATP synthesis. Mn++

is an essential nutrient, and ingestion from drinking water is assumed to represent a small proportion of total intake, typically contributing < 1% although this can rise to 20% depending on Mn

++ concentration in the water

(Loranger and Zayed, 1995). As Mn++

can occur in environment as pollutant in industrial and coal mine area. In certain geologic regions, long contact times between ground water and bedrock enriched in Mn

++

can lead to locally high levels in the water (U.S. Environmental Protection Agency (EPA, 2004). Sources of Mn

++ toxicity in litter of poultry include

feed additives, metabolic wastes, topical pesticides, and toxicants associated with the bedding material.

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53

95% excretion of Mn++

occur through biliary excretion, consequently any existing liver damage may decrease or delay its elimination and increase its amount in plasma (Ballatori, 2000). Mn

++ was found

cholestatic in cattle and rodents (Dahlstrom et al., 1991; Symonds and Hall, 1983). Mn

++ and bilirubin,

consistently induce intrahepatic cholestasis in rats and increase the cholesterol content in the bile canalicular membranes which line the bile duct. A recent study has determined that Mn

++ increases the

activity of 3-hydroxy-3-methylglutaryl coenzyme A, the rate limiting enzyme for cholesterol biosynthesis, and that bilirubin decreases cholesterol 7α- hydroxylase, which is important in the conversion of cholesterol into bile acids (Akoume, 2003). Modulation of these enzymes results in increased total cholesterol and decreased bile acid production. Acute liver toxicity was noted in steers after Mn

++

infusion into the duodenum for up to 30 h (Symonds and Hall, 1983). But the exact mechanism of Mn

++

induced toxicity in liver is not clear. Therefore, the present experiment was designed to identify the sub-acute toxicity of Mn

++ in poultry birds and to explore

the mechanism by which it produces liver toxicity.

MATERIALS AND METHODS

Sub acute toxicity of Mn++

in poultry:

Experimental animals: 18 healthy adult poultry birds of either sex(22-24 weeks ) were used and were maintained on balanced ration throughout the experiment. Drinking water was provided ad lib and the birds were allowed for acclimatization for seven days before start of experiment. Thereafter, all the birds were randomly grouped in to 3, possessing 6 birds in each.Group I (Control) were drenched with distilled water and group II and III were fed Mn++ @ 50 mg/kg and 100 mg/kg b.w. orally once daily for 28 days.

Parameters for observation: All the birds of Group I, II and III were subjected to the following studies during this experiment.

Total Protein

Prepration of tissue homogenate: The pieces of liver thus collected after the sacrifice of the experimental birds were accurately weighed. Frozen tissue samples were partially thawed and 1gm of tissue sample was weighed, 10% homogenate was prepared in ice-cold PBS with Remi-Homogenizer. The homogenate was centrifuged at 3000 rpm for 10 min and supernatant was used for biochemical estimations.

Total protein was measured on autoanalyzer by the method described by Doumas (1971). Protein reacts with Cu

++ in an alkaline medium to form a blue-violet

complex. Intensity of the colour formed during the fixed time is directly proportional to the amount of protein present in the sample. Protein level was expressed in mg/dl.

Serum concentrations of manganese: Mn

++ was quantitatively analysed on 0, 14 and day 29

during sub-acute toxicity study on GBC-902 Double Beam Atomic Absorption Spectrophotometer by the modified method of Kolmer et al. (1951). To 1 ml of serum and equal volume of concentrated HNO3 was mixed in the digestion tube. The samples were kept overnight at room temperature followed by digestion on low heat (70-80

0C) using heat block (digestion

bench), until the volume of sample reduced to about 0.5 ml. To this 1 ml of double acid mixture (3 part concentrated HNO3 and 1 part 70% HClO4) was added and low heat digestion continued until the digested samples became clear and emitted white fumes. As per need, the addition of 1 ml double acid mixture followed by low heat digestion continued until the digested samples became watery clear and emitted white fumes. Digested samples were diluted with 2 ml triple distilled water and filtered through Whatman filter paper No. 42. Repeated washing of digestion tube and filter paper were done by taking 0.5 ml triple distilled water. The filtrate was again diluted with triple distilled water to make the final volume to 5 ml.

Estimation of Mn++

concentration in liver: Two birds from each group were sacrificed for the estimation of Mn++ in liver tissue on day 29, was carried out with the help of GBC-902 Double Beam Atomic Absorption Spectrophotometer.

Digestion, extraction and preparation of samples:

Mn++

was quantitatively analyzed on day 29 in liver with the help of GBC-902 Double Beam Atomic Absorption Spectrophotometer by the method of Trolson (1969). Triturated tissue samples (1g) were taken in digestion tube and 5 ml concentrated HNO3 and 1 ml of concentrated H2SO4 were added and mixed well. The samples were kept overnight at room temperature followed by digestion on low heat (70-80

0 C) using heat block (digestion bench), until the

volume of sample reduced to about 1 ml. To this 3 ml of double acid mixture (3 part concentrated HNO3 and 1 part 70% HClO4) was added and low heat digestion continued until the digested samples became clear and emitted white fumes. Digested

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samples were diluted with 2 ml triple distilled water and filtered through Whatman filter paper No. 42. Repeated washing of digestion tube and filter paper were done by taking 0.5 ml triple distilled water. The filtrate was again diluted with triple distilled water to make the final volume to 10 ml.

Histochemical localization of ODFR generation: Site of Mn

++ induced generation of Oxygen Derived

Free Radicals(ODFR )was based on method of Kiernan (1999) and used for localization of dehydrogenases in the cells and Madesh and Balasubramanium (1998) used for assessment of Superoxide dismutase activity. It involves the principle that tetrazolium salt cannot be reduced by oxidases.

Reagents:

a) MTT (1.25mM): 2.58 mg MTT was dissolved in 5ml of D.W.

b) Phosphate buffer saline (PBS)- PBS was prepared by dissolving NaCl (8g) KCl (0.2g) KH2PO4(0.2g)and Na2HPO4 (0.94g)in 800ml distilled water. The pH was adjusted to 7.4 and the volume was made upto 1 lit with D.W.

c) Formal Buffer Saline Solution-It was prepared by dissolving 10ml of formaldehyde solution (40%) in 100ml of PBS.

d) Glycerine jelly.

Procedure:

a. Tissues from liver after necropsy of control and treatment birds were collected and slices of about 2-3mm thickness were made two from each tissue.

b. Slices from each tissue were incubated in MTT solution at 37

0c of

which one slice removed after 30 minute and another after 60 minute and kept in formal saline solution to terminate the reaction as well as to preserve the tissue for further tissue processing and cutting of section.

c. Tissues were washed in running tap water for 1h, dehydrated in ascending grades of alcohol (50%, 70% and 100%) for 20 minutes in each. Cleared in xylene

for 20 minutes and embedded in paraffin (60

0C) for 3h.

d. Blocks were made and sectioned of 5 µ thicknesses were cut and mounted on slides.

e. Paraffin was removed by dipping the sections in xylene for 1 minute, rinsed in water, mounted in glycerine jelly.

f. Sections were examined in light microscope as well as Phase contrast and Polarising microscopy.

Manganese localization: It was done as per method of Kiernan (1999). The details of the method is as below.

a) Paraffine cut sections were prepared as per Culling (1974), brought to deionized and distilled water.

b) 50 mg haematoxylene was dissolved in 1 ml of absolute alcohol which was made to 100 ml with deionised and diatilled water.

c) Sections brought to water were kept in haematoxylene solution for overnight.

d) Sections were given one change in 95% and two changes in 100% ethanol clear in xylene and mounted in synthetic resins mountaning medium.

e) Manganese gave blue to black reaction.

Histopathology: Formal saline fixed tissues from above mentioned organs were routinely processed, cut at 5mµ and stained with H&E stain as per method described by Culling (1974).

STATISTICAL ANALYSIS

Statistical analysis was carried out as per the formulae recommended by Snedecor and Cochran (1994). Quantitative data were analyzed, using the ANOVA. A value P <0.05 and P <0.01 were considered significant at 5% and 1% level respectively.

RESULTS

Total protein: Effect of sub-acute toxicity of Mn++

on total protein have been presented in table 1. The mean values of total protein in liver during the trial period did not show significant (P>0.05) difference. The mean values of protein in group I, group II and group III were 0.80±0.15, 0.85±0.00 and 0.57±0.11 g/dl respectively. It may be evident from table 1 that the total protein values in group ΙI and group ΙΙI were

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decreased on day 28 as compared to birds in group Ι. However, the above values of group ΙI and group ΙΙI did not differ statistically.

Table. 1: Effect of Mn++

on Total protein and concentration of Mn

++ in liver after daily single oral

dose (50 and 100 mg/kg) administration for 28 days in poultry.

Groups Group Ι(control) (n=6)

Group ΙΙ (n=6)

Group ΙΙI (n=6)

Total protein(g/dl)

0.80±0.15 0.85±0.00 0.57±0.11

Mn++

(ppm) 0.41±0.07 0.61±0.08 0.52±0.13

NS: Non Significant,

Blood Concentration (µg/ml): The mean concentrations of Mn

++ in blood have been

presented in table 2. The mean values of Mn++

concentrations on day 29 in group I, group II, and III were 0.05±0.005, 0.061±0.004 and 0.075±0.009 µg/ml respectively after daily oral administration at

the dose of 50 and 100 mg/kg for 28 days. There was increase in mean value of serum concentration of Mn

++ in treated groups.

Concentrations (ppm) of Mn++

in liver: The mean concentrations of Mn++ in liver after daily single dose oral administration of Mn++ for 28 day in poultry have been given in table 1. The mean concentrations of Mn++ in group II and group III were 0.61±0.08 and 0.52±0.13 ppm respectively on day 29. The mean value of Mn++ in group I birds was found to be 0.41±0.07 ppm. The concentrations of Mn++ in liver of treated groups were higher as compared to control group.

Histopathology: Microscopic examination of liver of group I failed to reveal any pathological alteration of significance. However, microscopically the liver of birds of group I fed with Mn

++ @ 50 mg/kg b.w.

showed varying degree of degenerative changes, mild periportal hepatitis along with venous congestion and sinusoidal dilation (fig 4).

Table. 2: Blood concentration (µg/ml) of Mn++

at different time intervals after daily single oral dose (50 and 100 mg/kg) oral administration in poultry.

Groups O Day 14 Day 29 Day Between days) F-value

Group Ι(control) (n=6)

0.048±0.004 0.045±0.006 0.05±0.005 0.22NS

Group І (n=6) 0.061±0.004 0.075±0.02 0.06±0.012 0.18NS

Group ІІ (n=6) A0.055±0.004

B0.085±0.007

AB0.075±0.009 4.33*

Between Grps) F-value

1.14NS

3.22 NS

1.82NS

NS= Non significant, *P<0.05, Mean under the same superscript in a row did not differ significantly.

Chronic Mn++

induced degenerative changes in this group varied from reversible to irreversible cellular injury. Reversible changes comprised of cellular swelling, granular and vaccuolar changes in cytoplasm as well as clumping and marginalization of chromatin materials in nuclei as evidenced by hepatocytomegaly, cytoplasmic eosinophilia, pyknotic fragmented nuclei, anucleosis and cytoplasmolysis with tendency of individuatization or even cell separation resembling that of apoptotic change. However, in some of hepatic cords such changes involved in a significant number of hepatocytes leading to hepatic cord disruption or atrophied hepatic cord.

One consistent and interesting finding with respect to hepatocellular necrosis was that of cytoplasmolysis characterized by presence of large moth eaten appearing clear vacuoles loss of cellular membrane

and presence of fragmented cytoplasm at cellular boundary when nuclei was found to remain intact indicating membrane damage as important site of action of Mn++. Sinusoids adjacent to swollen hepatocytes were compressed while those adjacent to cellular necrosis were dilated because of atrophied hepatic cords. Dilated sinusoids were appearing congested with presence of few erythrocytes in them. Moreover, the Kuffer cells were also hypertrophied and hyperactive with prominent and foamy cytoplasm.

Periportal hepatitis was characterized by mild infiltration of mononuclear cells in portal area with proliferation of bile duct epithelium (fig 7 and 8).

In birds fed with Mn++

@ 100 mg/kg b.w. hepatic changes were more developed and pronounced than group II. Birds of group III were exhibiting sever changes of irreversible injury characterized by

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hepatic necrosis incorporating comparatively large number of hepatocytes leading to complete disruption, disorganization or even collapse of hepatic architecture at number of places. At places, necrotic foci were also infiltered by inflammatory cells particularly mononuclear cells (fig 2,3,4,5 and 6).

Fig 1: Section of liver from a bird of group I showing periportal necrosis and mononuclear cells infiltration. (H&E × 400)

Fig 2: Section of liver from a bird of group II showing congestion and necrosis of hepatocytes. Hepatocytes are showing cytoplasmolysis, anucleosis and karyolysed nuclei. (H&E × 400)

Fig 3: Section of liver from a bird of group I showing granular and vacuolar changes in hepatocytes in focal area of necrosis with mild mononuclear cell infiltration. (H&E × 400)

Fig 4: Section of liver from a bird of group II showing hepatic necrosis, loss of lobular architecture with infiltration of mononuclear cells in area of hepatic necrosis. (H&E × 400)

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Fig 5: Section of liver from a bird of group II showing mononuclear cell infiltration with some hepatocytes undergone necrosis and degenerative changes. (H&E × 400)

Fig 6: Section of liver from a bird of group II showing mononuclear cells infiltration in area of necrosis. (H&E × 400)

Congestive changes were also sever incorporating central vein, portal vein and even sinusoids. Mononuclear infiltration and proliferation of bile duct epithelia was also pronounced and incorporated more number of portal areas.

Histochemical localization of Mn++

showed presence of Mn

++ in cytoplasm as blue to black granules or

clumps appearing as if Mn++

is mixed with aggregated or clumped cytoplamic constituents which were not discernable with H&E stain (fig 10, 11, and 12).

Positive reaction for Mn++

was found in granules which were dispersed in extracellular spaces after rupture of cells. Mn

++ positive granules and clumps

were more in cells undergoing degenerative changes. Moreover, Mn

++ granules were comparatively more in

number and size in hepatocytes of group III then that of group II. Though fewer in number, such granules were found in nuclei also. Moreover, cytoplasmic boundary and basement membranes were also giving positive reaction for Mn

++.

Phase contrast microscopy revealed heterogeneous nature of these granules. Moreover, the granules released from cells were exhibiting the feature of membrane or lipid bound bodies. Polarizing microscopy revealed these granules to be of amorphous in nature which indicated that Mn

++ might

be bind with cell constituents randomly.

Histochemical localization of ODFR exhibited very fine formazan granules in liver incubated for 30 minute. However, it was smaller as well as comparatively large sized in liver incubated for 1 h. Interesting finding was that the formazan granules were smaller and lesser than that Mn

++ bound

granules stained by histochemical staining. It suggest that not all Mn

++ molecules were active in generation

of ODFR rather some of them might be inactivated after binding with proteins appearing as large granules. Moreover, a large number of granules were found to be released from cells and get accumulated in extracellular spaces during clearing process of section by xylene. Phase contrast and Polarizing microscopic study revealed it to be of lipid and/or membrane bound debris (fig 9).

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Fig 7: Section of liver from a bird of group I showing single cell necrosis showing pyknosis and eosinophilic cytoplasm. (H&E × 400)

Fig 8: Section of liver from a bird of group I showing dilation of sinusoid, individualization of cells with features of cytoplasmolysis and loss of architectural arrangement. (H&E × 400)

Fig 9 : Section of liver from a bird of group II showing small formazan granules.

Fig 10: Section of liver from a bird of group I showing Mn

++ granules in hepatocytes. (Lilly & Fullmer ×400)

Fig 11: Section of liver from a bird of group I showing presence of Mn

++ granules in cytoplasm

of hepatocytes. (Lilly & Fullmer ×400)

Fig 12: Section of liver from a bird of group II showing comparatively more Mn++ granules in hepatocytes. (Lilly & Fullmer ×400)

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DISCUSSION

In chickens, manganese deficiency has been associated with perosis, thin-shelled eggs, poor hatchability, chondrodystrophy, and 60 ppm in the diet is the minimum recommended level (Puls, 1988). High dietary levels of MnCO3 in chickens (4779 ppm) causes decreased growth rate and increased mortality (Heller and Penquite, 1937), High levels of Mn

++ in drinking water (Bouchard

et al., 2007) and

infant formula with high Mn++

content (Collipp et al.,

1983) have been considered as possible sources of exposure.

The concentrations of Mn++

in the liver were found to be higher in group II and group III as compared to group I birds. However, the above difference was not significant (P>0.05). The decrease in total protein value (table 1) indicates that there was liver dysfunction. On the basis of results obtained we can predict that the above higher level of Mn

++ in liver

could damage the liver tissue. This could further be evident from the work of Berta et al. (2004) in which he found a significant (P < 0.05) increase in accumulation of the Mn

++ in the liver with

supplements of 60 and 240 ppm of MnO and Mn fumarate in case of broiler chicks.

Liver showed a dose dependent variation in degree of changes of reversible hepatocellular injury characterized by cellular swelling, granular and vacuolar changes in mildly affected cells. It also showed apoptic and necrotic changes characterized by cytoplasmolysis, cytoplasmic eosinophillia, pyknotic karyorrhactic and karyolised nuclei. Along with hepatocellular changes, liver also exhibited focal to multifocal areas of haemorrhage and infiltrative changes (fig 1,2 and 3).

Hepatocellular necrosis has also been reported in rats, rabbit, dogs and humans Wit(Misselwitz et al., 1995; zleben et al., 1968 and WHO, 1981). Such changes have also been due to Mn

++-complexed

immaging agent (Lim et al., 1991) and following receipt of parentral nutrition in human (Mehta and Reilly, 1990). After oral or parentral administration Mn

++ is rapidly eliminated from blood, concentrated

primarily in liver and excreted in the bile (Grant et al., 1993; Klaassen, 1974; Malecki et al., 1995; Saini et al., 1995 and Saric, 1986).The increased susceptibility of liver to Mn

++ toxicity appears to be

associated with greater accumulation of Mn++

in liver after oral administration.Mn

++ is alleged to exert

cellular toxicity via a number of mechanism including a direct or indirect formation of ROS (Ali et al., 1995)

and direct oxidation of biological molecules. However, the exact intracellular sites of initiation, propagation and damage of biological molecules through this mechanism are far from clear. Moreover, the precise pathways and sequence of events that lead to cellular injury due to Mn

++ is also not

understood. Directly Mn++

induced generation of ODFRs is not known and requires further study in depth.It has suggested that cytotoxic effect of Mn

++

may be due to its effect on cell membrane via oxidation of divalent Mn

++ to trivalent Mn

++ by oxygen

radicals (Misselwitz et al., 1995). It is alleged that Mn

++ accumulates in mitochondria disrupts oxidative

phosphorylation and increase the production of ROS with subsequent lipid peroxidation (Gunter et at., 2006; Gavin et al., 1992, 1999).A recent report of Kalia el al., 2008 showed that upon fractionation of neuronal cells, labeled Mn

++ was found primarily in

nuclei with virtually rare in the mitochondrial fraction and therefore mitochondria may play insignificant role in sub cellular distribution of Mn

++ and has raised

debate of intracellular localization of Mn++

. Surprisingly, despite such controversy exact localization of Mn

++ in intact cells particularly injured

due to Mn++

toxicity does not appear in literatures. This warranted intracellular localization of Mn

++ in cell

and site of generation of free radical, there after particularly in Mn

++ induced injured cell. Keeping

these facts in mind the present study incorporated localization of Mn++ in hepatocytes in order to throw more insight on mechanism of cellular injury due to Mn++ toxicity.Localization of Mn

++ showed presence

of Mn++

in cytoplasmic granules characteristic of granular changes induced due to Mn

++ toxicity in

hepatocytes (fig 10,11 and 12). Such positive reaction was found to label cytoplasmic membrans also. Increased number of heterochromatins in nuclei were also found to show presence of Mn

++.

Phase contrast microscopy followed by polarizing microscopy suggested these granules to be almost amorphous in nature (fig 9). It indicated that Mn

++

molecules are not arranged in crystalline from rather they are randomly and irregularly bound with degenerated cellular components aggregated in form of cytoplasmic granules appearing due to Mn

++

toxicity.Granular changes in the cytoplasm is an after math of clumping and aggregation of soluble proteins due to change in their conformational structure, protein- protein sulphydryl linkages and protein fragmentation along with binding of lipid due to exposed lipophilic sites of protein (Cotran, 1999). Thus, presence of Mn

++ in cytoplasmic granules

suggest their involvement in these degenerative

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changes.Though the mechanism of cellular damage is speculated to involve either direct or indirect generation of free radicals or direct binding with the biomolecules. But which mechanism is predominant at intracellular site is not clear. So, this study was also aimed at ascertain the exact intracellular site of Mn

++ induced generation of free radicals involved in

cellular injury.

This experiment incorporated modification of previous methods of Culling (1974) and Kiernan (1999) using tetrazolium salt to locate the activity of dehydrogenases, oxidases and superoxides. The modification is based on the principle that terazolium salt, an artificial hydrogen acceptor for different dehydrogenases are changed by reduction into insoluble coloured formazan. The importance of this artificial electron acceptor lies in the fact that the flow of electron may be interrupted by the introduction of an artificial electron acceptor with an oxidation-reduction potential intermediate between those of any two of the member of the electron transport chain (Kiernan, 1999). Tetrazolium can be reduced by oxidoreductase using coenzyme NAD

+ and NADP

+

as well as FAD as prosthetic group. But cannot be reduced by oxidoreductase using molecular oxygen as electron acceptor because its oxidation-reduction potential does not lie intermediate between them (Kiernan, 1999). However, Superoxide ion, 02

- a free

radical is able to reduce tetrazolium salts directly because favourable redox potential.Our modification used blocking of reduction of tetrazolium by intracellular dehydrogenases by not providing substrates for them in incubation medium as used in the method of Culling (1974) to locate the activity of dehydrogenases. Further, in the original method cobaltous chloride with tetrazolium was used which after being reduced has leading to formation of cobalt chelate of black crystal formazan (Kiernan, 1999). Mn

++ can also form chelates of formazan if

tetrazolium is reduced in presence of manganese ion.Such chelate formation is more favorable if Mn

++

is itself involved in generation of O2- causing

reduction of tetrazolium salt. If Mn++

is involved in generation of superoxide, it will lead to formation of Formazan crystal if tissue collected were immediately incubated with tetrazolium in suitable medium for this reaction. This assumption was used in designing a histochemical staining procedure for ascertaing the role of Mn

++ induced free radical generation in

cytotoxicity and localization of intracellular sites of their activity. The reaction of this histochemical staning come out as expected lines was formation of black formazan crystals in Mn

++ containing cells

which were further confirmed by phase contrast microscopy as well as polarizing microscopy during preliminary experimental trials. Thereafter, a protocol was established which was used in this study as mentioned in material and methods.

Staining of liver section with this protocol showed presence of fine formation granules distributed in the cytoplasm of hepatocytes of Mn

++ treated birds (fig

9). Granules were invariably of smaller size in tissues incubated for 30 minute. It was of small as well as large sized in tissues incubated for 1h. Mitochondria is the main site of generation of Mn

++ induced ODFR

as well as it is the main site of Mn++

accumulation. A longer reaction period for formazan formation cause damage of mitochondria due to increased osmotic pressure and cause diffusion of granules in vicinity of mitochondria appearing as large granules, if they are not protected by polyvinyl alcohol. Thus it could be assumed that large sized granules were site of generation of O2

- in mitochondria while small sized

granules were site of extra mitochondrial O2-

generation. However, this warrants further exploration and confirmation which will be of great help in understanding the molecular mechanism underlying Mn

++ toxicity. However, taking these

assumptions into consideration, there was comparatively more number of both extramitochondrial and intramitochondrial granules in liver of 100 mg/kg b.w. treated birds, while extra mitochondrial granules were lower in birds treated with Mn++ @ 50 mg/ Kg b.w.It suggested that at higher dose of Mn

++ there was generation of ODFR in

mitochondria as well as in extramitochondrial cytoplasm. While at lower dose rate, it was mostly restricted in mitochondria only. Upon entry in the cells, mitochondria is a primary and initial site of Mn

++

accumulation. Mitochondrial sequesterization does not represent simple binding but weak binding with a steady control by influx and efflux of Mn

++ ions

(Gunter and Pfeiffer, 1990). It is influxed by mitochondrial Ca

2+uniporter, primarily energized by

internal negative memberane potential and effluxed by the Na

+ independent mechanism primarily

energized by the pH gradient. Both are maintained by energy dependent proton pump across inner mitochondrial membrane. Mn++ decreases energy metabolism in vivo and in vitro including decreased in activity of mitochondrial enzyme in membrane potentials and increases ROS generation and subsequent lipid peroxidation.Thus energy depletion, altered membrane potential and intra mitochondrial generation of ODFR might be causing desequesterization of Mn

++ in extramitochondrial sites

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61

of cytoplasma and generation of ODFR at that site might be due to direct oxidation of O2

- or other

biological molecules there.Chelates of formazan are lipid soluble (Kiernan, 1999) and can be localized in lipids, xylene or acetone extracts and some of the cytoplasmic lipids in which formazan may remain accumulated (Kiernen, 1999).

When paraffin cut sections of liver of Mn++

treated birds were dipped for 1 minute in xylene during processing a large number of lipid or membrane bound bodies containing black or blue deposits were found to migrate from cell and accumulate in extracellular spaces in slides. Phase contrast microscopy and Polarizing microscopic examination also revealed that there are lipid or membrane bound formazan containing granules which have been extracted from the cell during processing of the sections in xylene or acetone leaving areas appearing as cytoplasmic vaccules. Feature of cytoplasmolysis which were not found to be present and migrate in control sections. It could be easily interpreted that changes of cytoplasmolysis are an after math of removal of protein, tissue debries formed due to degenerative change during processing of the paraffin section in xylene or acetone. Presence of formazn granules in lipid or membrane bound bodies of tissue debris revealed that such granules were formed in close vicinity of sites where degenerative change were going on which indicated the presence of Mn

++ at that site and

their generation of free radicals also there. It suggested involvement of Mn

++ induced ODFR

generation in pathogenesis of cellular changes in cytoplasm. Further exploration of these extracted lipid or membrane bound granules may throw more insight on mechanism of vacuolar degeneration i.e. due to mitochondrial damage or due to lipid peroxidation of membrane.Thus the finding led to the conclusion that Mn

++ is primarily accumulated in mitochondria. It is

sequesterized there by an energy dependent process. Entry of Mn

++ in cells above level of

sequesterizing capacity may cause disruption of oxidative phosphorylation and damage due to generation of ODFR exhibiting changes of reversible cellular injury in light microscope.

Such disruption is reversible till there is no profound damage of inner mitochondrial membrane. However, depletion of mitochondrial energy may further cause desequesterization of Mn

++ if increased level of Mn

++

persisted or further increased. This in turn perpetuates vicious cycle of desequesterization of Mn

++ and Mn

++ induced oxidative damage leading to

profound organellar and cellular membrane damage including inner mitochondrial membrane brings inevitable death of cells revealing changes of cell necrosis.

Acknowledgements

Authors acknowledge I.C.A.R.,for providing the fund under Outreach Programme on “Monitoring of Drug Residue & Environmental Pollutants”for this study.

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