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lemental alteration, iron overloading and metallothionein induction inxperimental hepatocarcinogenesis: A free radical-mediated process?
ebadutta Mishra ∗, Mathummal Sudarshan, Anindita ChakrabortyGC-DAE Consortium for Scientific Research, Kolkata Centre, III/LB-8, Salt Lake, Kolkata 700098, India
r t i c l e i n f o
rticle history:eceived 26 October 2010eceived in revised form 17 February 2011ccepted 21 February 2011vailable online 3 March 2011
eywords:
a b s t r a c t
The azo-dye p-dimethylaminoazobenzene (p-DAB) is a potential tumor initiator in rodents, but the under-lying mechanism is not clear. Following chronic feeding of the carcinogen for 3, 5 and 7 weeks, traceelemental status, free radical generation, oxidative damage, antioxidant profile were measured in maleand female swiss albino mice. The feeding resulted in iron accumulation in male mice liver. No increasein iron level was observed in similarly exposed female mice. The results of this study suggest that p-DAB-induced iron accumulation in male mice with concomitant production of oxidative free radicals is an
early event in the hepatocarcinogenic initiation. This occurs selectively in male mice and affects eitherdirectly or indirectly in development of chemically induced liver neoplasia. Again, that upregulation ofmetallothionein (MT) expression in association with increased free radical generation was demonstratedin male mice. Alteration of copper (Cu) and zinc (Zn) levels are described in the light of antioxidant pro-file in liver tissue. The current results thus provide evidence in support of iron accumulations producingoxidative damage, and enhanced metallothionein expression as possible contributors in the mode ofaction of p-DAB induced hepatocarcinogenesis.
. Introduction
Chronic injury due to carcinogenic insult deregulates cellularomeostasis and induces a number of alterations leading to disrup-ion of cellular processes such as cell cycle and apoptosis, drivingo carcinogenesis.
Trace elements regulate enzymatic activities, immunologicaleactions, physiological mechanisms and carcinogenesis by act-ng both as essential and toxic components in body metabolismGoldhaber, 2003; Mertz, 1998). Alterations in metal levelsesulting in deregulation of homeostatic activities have beenemonstrated in animal models (Hayakawa et al., 1997).
Toxic and carcinogenic effects of exposure to certain transitionaletals are well known. Metals, such as zinc, iron, copper, cobalt
nd manganese in coordination with each other participate in theontrol of various metabolic and signaling pathways. Redox prop-rties of these metals are such that they are capable of escaping
ut of the homeostatic control mechanisms. Breakdown of theseechanisms can lead to the metal binding to protein sites other
han those tailored for that purpose or displacement of other met-ls from their natural binding sites. Further, interaction of these
metals with nuclear proteins and DNA causes oxidative deterio-ration of biological macromolecules (Valko et al., 2005). Iron is animportant redox active element, the carcinogenicity of which is stillunder debate unlike arsenic, chromium or nickel.
Chemically induced carcinogenesis is a multistep processcharacterized by at least three steps: initiation, promotion andprogression. There are several models available to study themechanisms of development of liver cancer in vivo. Hepato-carcinogenesis induced by various procarcinogens like para-dimethylaminoazobenzine (p-DAB), diethylnitrosamine (DEN),3′-methyl-4-dimethylnitrosamine (3′-MeDAB), etc. is an intricateprocess and provides a favorable model that facilitates study ofthe entire process of chemical carcinogenesis (Farber, 1980). Acommon requirement of all the chemical carcinogens is the acti-vation of the chemicals to reactive electrophiles, also referred toas ultimate carcinogen, which becomes covalently bound to DNAto cause mutation and cancer (Guengerich, 2001). The azo-dye p-dimethylaminoazobenzene (p-DAB), used as a coloring agent forpolishes and soap is a slow acting carcinogen (Ling and Foster,1980), which only initiates liver carcinogenesis without any promo-tional effects, but the mechanism of carcinogenesis of this chemical
is obscure.
Current research has postulated the involvement of endoge-nously produced reactive oxygen species (ROS) in activation ofcarcinogens to an electrophilic DNA-damaging moiety, which isan essential requirement in the initiation step, and can also bind
irectly to bring about changes in DNA. Redox active metals likee, Cu etc. are most potent oxidants which evoke oxidative stressia production of oxygen-derived free radicals such as superoxidesO2−), hydroxyl radical (OH−) and intermediate oxygen-reductionroducts such as hydrogen peroxide (H2O2) (Perera et al., 1987).linkage between an increase in cellular reactive oxygen radicals
nd the pathogenesis of several chronic diseases including cancer isell established (Toyokuni, 1996). Mutagenesis by ROS could con-
ribute to the initiation of cancer, in addition to being important inromotion and progression phases.
The cellular requirement of essential metals, their homeostaticontrol, and biological effects are controlled by a number of spe-ific metal binding proteins. These metalloproteins are involved inhe storage, transport, and biological properties of many metalsCherian, 1994).
Metallothionein (MT), a trace metal-binding protein involvedn the maintenance of homeostasis of essential trace metals suchs zinc (Zn) and copper (Cu) (Bremner and Beattie, 1990; Cousins,985; Margoshes and Vallee, 1957) is now a days well recog-ized as one of the stress proteins induced by various heavyetals and oxidative stress (Hamer, 1986). It functions in the
ytosolic compartment as a chelator of various heavy metal ions,uch as zinc, copper and cadmium and as a scavenger of reac-ive oxygen species both in vivo and in vitro. The protectiveole of MT against carcinogenesis has been documented, but thenderlying mechanism remains unclear. As an antioxidant, MTay delay the promotion step of carcinogenesis (Kondo et al.,
999).Short-term (3–7 weeks) administration of p-DAB, following Solt
nd Farber protocol of chemical carcinogenesis resulted in markedlterations in several biochemical parameters and trace elements.epatic accumulation of iron has been associated with inductionf liver cancer. However, the cause of elevated iron stores duringxperimental liver carcinogenesis is unclear (Cragg et al., 1998;ischer et al., 2002). The present study is designed to probe intohe precise role played by trace metals, particularly iron in car-inogenesis. The basic objective is to understand if trace elementslay role in the mechanism of carcinogenic action of the chem-
cal, used in the present study. Effect of the generated ROS onntegrity of RBC, MT induction and antioxidant profile has been
easured in the animal subject. This paper provides data show-ng that, variations in the hepatic level of iron and metallothioneinMT), the metal-binding protein, starting from the early stage of car-inogenesis, may be useful targets in the detection of early-stagealignancy.
. Materials and methods
.1. Chemicals
p-DAB and collagenase, BSA were purchased from Sigma Chemical Co. (St. Louis,O). Rabbit polyclonal MT antibody (FL-61) antirabbit horse IgG and goat anti
abbit-IgG-FITC were purchased from Santa Cruz Biotechnology, Inc., USA.
.2. Animals
Inbred strain of Swiss albino mice (Mus musculus), 6–8 weeks old, weighing4–27 g were acclimated for 2 weeks in well aerated, pathogen free conditions withree access to water and diet prior to p-DAB treatment. The recommendations ofhe NIH guideline for the Care and Use of Laboratory Animals were followed for the
aintenance, treatment and sacrifice of the animals used in this study (NRC, 1985).
.3. Experimental design
The animals were divided into six treatment groups [10 mice per group]. Threeroups of mice received p-DAB at a dose of 0.05 mg/kg body weight via daily gavagesingle dose per day) for 3, 5 and 7 weeks, respectively (Biswas and Khuda-Bukhsh,005). Rest three groups of mice were kept as control. Body weights of the miceere noted at regular intervals.
tters 203 (2011) 40–47 41
At the end of 3rd, 5th and 7th week, the animals were weighed and sacrificed byasphyxiation. The liver, kidney and spleen were immediately removed and weighed.Livers was perfused with 1× PBS, removed and weighed. A portion of liver wasimmediately processed for elemental analysis.
2.4. Fractionation of liver
The liver was promptly removed after sacrifice, minced, washed to remove bloodand then homogenized in 1.5% KCl using Teflon pestle. Centrifugation at successivelyhigher speeds yielded the following fractions: crude nuclear fraction at 1000 × g for10 min; heavy mitochondria at 3000 × g for 10 min; light mitochondria at 20,000 × gfor 20 min; and microsomes at 144,000 × g for 90 min. The final supernatant was thecytosolic fraction (Okado-Matsumoto and Fridovich, 2001).
2.5. Hematocrit
Blood was collected by cardiac puncture and collected in micro-centrifuge tubes.The collected blood was then centrifuged at 10,000 rpm in a Hitachi made high-speed refrigerated centrifuge (model CR21E) at 4 ◦C for 10 min and the percenthematocrit determined (percent red blood cells in total blood volume).
Plasma was separated, made into aliquots and immediately transferred to−80 ◦Cuntil analysis.
2.6. Measurement of intracellular oxidative stress
Intracellular free radicals were detected using dihydrorhodamine 123 [CaymanChemical Co.]. Dihydrorhodamine 123 is an uncharged, non-fluorescent product thatactively diffuses across most cell membranes where it is oxidized to rhodamine 123and locates in the mitochondria (Agadjanyan et al., 2003). To diluted whole blood,123 dihydrorhodamine was added at 5 �g/ml from 2.5 mg/ml stock in ethanol. Flu-orescence intensity was measured using PerkinElmer luminescence spectrometer[Excitation, 490 nm; emission, 530 nm].
2.7. Atomic force microscopy of RBC
The blood cells collected from heart of anaesthetized mice administered withchemical carcinogen was fixed in 2.5% glutaraldehyde and the blood was dropped ona clean glass surface to make a thin uniform smear of blood cells and then scannedwith Nanoscope IV (Vecco Instruments Co.). The images were obtained in normaltopographic mode as well as Lateral force mode. The same was done from blood ofcontrol group animals.
2.8. Elemental analysis
Samples of liver and its sub-cellular fractions, kidney, serum and RBC [from eachgroup] were lyophilized at −85 ◦C in a freeze-dryer and then crushed to powder bybrittle fracture technique. These were then palletized into thick targets of 8 mmdiameter and 1 mm thickness using Pelletizer at 110 kg/cm. pressure. In each groupconsisting of 10 animals, a minimum of three pallets per animal was considered inorder to decrease the sampling error. All samples were studied without any chemicaltreatment.
2.9. Instrumentation
Elemental analysis was done by energy dispersive X-ray fluorescence spec-troscopy (Jordan Valley-EX-3600) technique. EDXRF study was carried out at 50 kVvoltage and 1 mA current using Si(Li) detector having energy resolution of 143 eV at5.9 keV. The quantitative analysis of elements ranging from Z = 13 (Al), to Z = 82 (Pb)was done using inbuilt EXWIN software (Carvalho and Marques, 2001).
2.10. Test for lipid peroxidation
Quantitative determination of total protein of the supernatant obtained aftercentrifugation at 3000 × g was done following the method of Lowry et al. (1951)and lipid peroxidation was assessed by measuring thiobarbituric acid reactivesubstances (TBARS) at 535 following the method of Buege and Aust (1978). The mal-ondialdehyde (MDA) concentration of the sample was calculated using extinctioncoefficient
∑= 1.56 × 105 M−1 cm−1.
2.11. Test for antioxidant enzymes
The supernatant as mentioned above was used for enzyme assays. Catalase activ-ity was measured as the rate of decomposition of H2O2 at 240 nm following themethod of Aebi (1984). Rate of NADH oxidation as a measure of SOD level was
studied following the method of Paoletti et al. (1986).
2.12. Isolation of hepatocytes
Hepatocytes were prepared by two-step collagenase perfusion method (Staubliand Boelsterli, 1998). After mechanical disruption of liver capsule, the liver cells
42 D. Mishra et al. / Toxicology Letters 203 (2011) 40–47
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ig. 1. Elemental Concentration in whole liver. Statistically different from the con-rol; *P = < 0.05, **P = < 0.005, ***P = < 0.001, NS: not significant.
ere collected in Hanks’ balanced salt solution (HBSS) and serially filtered in 80-nd 60-mesh after two rounds of sedimentation. The cell viability was assesses byrypan blue and cell number was adjusted to 0.5–2 × 106 viable cells/ml.
.13. Intracellular staining and FACS analysis
Cells collected by centrifugation were washed and then resuspended in 1 ml ofBS, fixed in 2% formaldehyde for 10 min at 37 ◦C and then permeabilized in ice-coldethanol. After blocking in BSA, rabbit polyclonal MT antibody (FL-61, Santa Cruz
iotechnology, Inc., USA) was added at 1:100 dilutions and incubated for 60 min.ells were then rinsed and resuspended in goat anti rabbit-IgG-FITC [Santa Cruziotechnology, Inc. USA] and Flow cytometry was performed on a FACSCalibur usingellQuest software (Becton-Dickinson, San Jose, CA).
.14. MT localization study by immunoelectron microscopy
L.R. white sections were blocked in fish gelatin and then incubated in rabbitolyclonal MT primary antibody in tris-buffered saline [1:100] over night at 4 ◦C,ollowed by gold-labeled secondary antibody [1:20] and visualized using FEI Philips
orgagni 268D transmission electron microscope.
.15. Aconitase assay
The aconitase activity was measured in the cytosolic extract of the liveromogenate spectrophotometrically at 340 nm by monitoring rate of NADH reduc-ion in presence of sodium citrate and isocitrate dehydrogenase following the
ethod as described by Drapier and Hibbs (1996).
.16. Statistical analysis
Student–Newman–Keuls (ANOVA) test was used to assess the degree of signif-cance. Data were presented as Mean ± SD. Differences were judged as statisticallyignificant for P ≤ 0.05.
. Results and discussion
Results of the present study reflect significant alterations inlemental profile in the liver of male mice following p-DAB admin-stration. Among the elements qualitatively and quantitativelyvaluated, increased in concentrations of Fe, V, Cr and Ni wereoted when compared to the untreated animal group considereds the normal counterpart (Fig. 1) indicating these elements asefined markers for the onset of liver carcinogenesis. Depletion of
ome elements like Zn, Cu was observed in the successive phasesf initiation in liver tissue. Fe, Zn and Cu levels were decreased inerum of treated group, with no detectable elemental alterationn RBC. Concentration of Fe, Cu and Zn were increased in kidneyf the treated group. This reverse trend of alteration in profile of
Fig. 2. Elemental Concentration in liver (a) Mitochondria and (b) Nucleus. Statisti-cally different from the control; *P = < 0.05, **P = < 0.001, NS: not significant.
some essential elements, as mentioned above, possibly indicatescoordinated role of these trace elements in different metabolicpathways (Storey and Greger, 1987). It has been shown earlierthat onset of metabolic deregulation caused by any carcinogenicinsult gets first reflected in the liver tissue of the host (Bannaschet al., 2003). The present observation is in line with the earlierreviews (Oliveira et al., 2007) establishing inter-individual differ-ence in the metabolism of carcinogens. Possibly this might inferto the actual involvement of specific elements in mechanism ofcarcinogenesis.
Overloading of Fe, presumably arising from red blood cellhemolysis, along with its increased concentration in mitochon-dria and nuclear fractions (Fig. 2) relative to that in control animalclearly reflects role of iron in initiation event of p-DAB inducedcarcinogenesis. There was no detectable change in cytosolic ironcontent.
No such variation in elemental profile was observed either inwhole liver or its sub-cellular parts of similarly exposed femalemice, indicating that female Swiss albino mice are refractory to pre-
neoplastic alterations under conditions described in the currentpaper and were thus excluded from further experiments detailedhere. The lack of hepatocarcinogenic activity of the dye in femalemice is consistent with the refractoriness of female mice, as con-
ogy Letters 203 (2011) 40–47 43
trwrhadpdnePotiNlofits1esgTIteiiamusiitm
I(hoecoteiid
i
Fig. 3. Intracellular oxidative stress profiling in whole blood as measured by oxida-
TB
D. Mishra et al. / Toxicol
rasted to their male littermates, to other hepatic carcinogenseported by earlier workers (Ward et al., 1996). Among the factorshich may contribute to the sensitivity of male mice are the rapid
ate of liver growth at the time of exposure to the carcinogen, theormonal environment of the liver cells in the adult male animals,nd undefined genetic factors. Feeding of carcinogenic azo dye pro-uces liver damage followed by regeneration of parenchymal cells,roliferation of bile ducts and connective tissue, and at later stagesevelopment of tumors from liver parenchyma that end up witheoplastic characteristics (Kitagawa and Sugano, 1977; Manjeswart al., 1994; Aydinlik et al., 2001; Biswas and Khuda-Bukhsh, 2004;athak and Khuda-Bukhsh, 2007). It has been observed in somether investigations from our group that long-term administra-ion (for more than 43 weeks) of p-DAB has resulted in tumorsn mice (unpublished data). This finds support from the report ofesnow et al. (1987) who mentioned chronic feeding for a very
ong time 45–61 weeks induces tumors in rodents. However webserved such incidence only in case of male mice. Similar to ourndings, reports from IARC have documented carcinogenic poten-ial of p-DAB mostly in male rodents where female mice did nothow tumor induction under similar conditions of exposure (IARC,975). This might be due to the fact that female metabolism ofxogenous agents differs markedly from that of males as has beeneen in case of gender specific difference in promotion of tumori-enesis in rats by phenobarbital administration (Nims et al., 1993).hese differences may be directly or indirectly hormonally driven.t appears that this apparent lack of responsiveness is due moreo experimental design rather than to physiologic response (Wardt al., 1996). As such, female mice showed hepatocellular tumorncidences close to those of male mice if allowed to live longer orf chemical doses were adjusted. Thus in the present investigationbsence of significant changes in the elemental profile in femaleice during the initiation phase of carcinogenic insult prompted
s to exclude female mice from further experimental parameterstudied in the present investigation. As such it is plausible thatnitiation of the physiological alterations induced by carcinogenicnsult apparently provide the necessary promoting stimulus for fur-her transformation towards neoplastic development in the male
ice.The liver is the main organ for iron storage and metabolism.
t is evidenced that iron overload is a risk factor for liver cancerBonkovsky, 1991; Huang, 2003). Excessive accumulation of iron inepatocytes causes hepatocellular injury, which leads to the devel-pment of fibrosis, cirrhosis and hepatoma (Britton, 1996; Kangt al., 1998; Niederau et al., 1985). Hepatic accumulation of ironan produce reactive oxygen species leading to lipid peroxidation,xidation of proteins and oxidative DNA damage, associated withhe induction of liver cancer (Kasai and Nishimura, 1984; Klaunigt al., 1998; Wiseman and Halliwell, 1996). In the present study an
ncrease in hemolysis as measured by the decrease in hematocritn addition to increase in relative spleen weight was observed in aose dependent manner (Table 1).
These findings prompt us to propose that the increase inron deposition via hemolysis results in an increase in liver
able 1ody weights, hematocrit and relative spleen weights following p-DAB treatment for 3, 5
* Relative spleen weight = [(spleen wt./body wt.) × 100].a No significant differences (P > 0.05) between p-DAB-treated and control groups.b Significant differences (P < 0.05) between p-DAB-treated and control groups.
tion of 123 dihydrorhodamine. The emission spectra at 530 nm with excitation at490 nm of blood obtained from control and p-DAB treated mice as recorded using aPerkinElmer fluorimeter.
oxidative stress (via the generation of hydroxyl radicals by Fen-ton reactions), and ultimately leads to the observed neoplasticresponse in male mouse liver. The observed data of the presentinvestigation designate and further establish role of iron incarcinogenesis.
Our results show enhanced 123-dihydro-rhodamine oxidationin blood of the p-DAB treated group than that in the control group asdetermined by significant increase in fluorescence intensity (Fig. 3).Though there is not enough cellular evidence linking p-DAB directlyto oxidative mechanism of cellular transformation, present datareflect p-DAB as a potential inducer of free radicals and reactiveoxygen species (ROS).
ROSs are known to induce peroxidative damage to the mem-branes thereby resulting in loss of membrane integrity (Cerutti,1985). Such damage due to carcinogenic insult has been reflectedin RBC of the carcinogen treated mice when observed under AFM(Fig. 4). Characteristic blebs and holes in the RBC were observed.Thus, the observed increase in oxidative stress due to p-DAB treat-ment may directly be linked to oxidative mechanism of cellulartransformation involving iron metabolism.
Since the body does not have a mechanism to excrete out excessiron that is entering and iron homeostasis is strictly maintainedat the level of intestinal absorption, excess body iron stores maybe detrimental to the cells by both catalyzing generation of cyto-toxic reactive oxygen species and by acting as limiting nutrientto the growth and replication of cancer cells. Additionally, incre-
ment in levels of Cr and Ni in association with depletion in zinclevel as observed in the present data reflects possibly more stressgeneration leading towards further metabolic deregulation. Thisis in good conformity with the available reports where Cr and Ni
and 7 weeks.
t (%) Relative spleen weight*
p-DAB Control p-DAB
43.4 ± 1.6b 0.39 ± 0.04 0.44 ± 0.09b
42.8 ± 0.8b 0.35 ± 0.07 0.41 ± 0.07b
42.1 ± 1.0b 0.28 ± 0.04 0.40 ± 0.07b
44 D. Mishra et al. / Toxicology Letters 203 (2011) 40–47
Fig. 4. AFM images showing smooth surface topography of a red blood cell from a normal untreated mice (a) and the corresponding lateral force microscopic (LFM) imageobtained by taking into account the horizontal component of the force (b) compared to AFM images of red blood cell from p-DAB treated mice (c) and the corresponding LFMi . The( rred to
a(zrot1
wp
ttawi
mage (d) showing bleb formation. Area scanned 8 �m × 8 �m, height (Z) range 3 �mFor interpretation of the references to color in this figure legend, the reader is refe
re shown to be carcinogenic to humans or experimental animalsPoirier, 1996). It has also been established that low intracellularinc induces oxidative damage in addition to interference with DNAepair via disruption of p53 (Emily and Ames, 2002). Induction ofxy- and nitro-radicals due to such overloading has been proposedo have indirect role in pre-neoplastic alterations (Klaunig et al.,998).
The data presented here shows that hepatic lipid peroxidationas significantly increased during all p-DAB feeding intervals com-ared to control animals (Fig. 5).
The observed enhancement of ROS production complies with
he noted alterations in activities of catalase and superoxide dismu-ase (Fig. 6), which showed slight increase in activity at 3rd weeknd then decreased with subsequent treatment when comparedith the control groups. This confirms profound effect of free rad-
cal generation by p-DAB on antioxidant status of the mice. Thus
colors indicate the height of the surface – dark brown being low, yellow being highthe web version of the article.).
the carcinogen p-DAB effectively induces alterations in elementalconstituents and oxidant–antioxidant profile in the administeredanimals.
However imbalance and/or perturbations with regard tohomeostasis of essential metals and subsequent metabolic co-ordinations are controlled by a number of specific metal bindingproteins. These metalloproteins are involved in the storage, trans-port, and biological properties of many metals (Cherian, 1994).Metallothionein (MT) is one such protein. It belongs to a familyof low molecular weight, cysteine-rich proteins primarily involvedin metal homeostasis and detoxification and is also one of the
stress proteins induced by various heavy metals and oxidativestress (Kagi and Shaffer, 1988). It functions in the cytosolic com-partment as a chelator of various heavy metal ions, such as zinc,copper and cadmium and as a scavenger of reactive oxygen speciesboth in vivo and in vitro. Although MT induction by Fe has not
D. Mishra et al. / Toxicology Letters 203 (2011) 40–47 45
Fig. 5. Lipid peroxidation in liver of control and treated mice at different feedingintervals. *P = < 0.001.
Table 2Mean fluorescence intensity (MFI) of FITC labeled MT expression as calculated byBD-CellQuest software.
Sample Events Geo. mean
Negative control 20,000 5.67Labeled control 20,000 124.48
btVMbM1ictofliccnicip
FtFc
MT induction after 3 weeks of p-DAB treatment 20,000 301.86MT induction after 5 weeks of p-DAB treatment 20,000 338.56
een well established, iron overload is documented to cause oxida-ive stress in experimental animals (Fleet et al., 1990; Good andasak, 1986; Yasutake and Hirayama, 2004). The protective role ofT against carcinogenesis has been documented (Hamer, 1986),
ut the underlying mechanism remains unclear. As an antioxidant,T may delay the promotion step of carcinogenesis (Kondo et al.,
999). In the present study increase in oxidative stress resultedn significant enhancement of MT expression from 3rd week ofarcinogen treatment (Fig. 7), suggesting its involvement in protec-ion against iron induced oxidative stress in early initiation phasef carcinogenesis. Increase in MT expression is reflected in theow cytometric analysis of MT showing its significant increase
n mean fluorescence intensity (Table 2) in the treated group inomparison to that observed in case of the control group. Further,ytosolic localization of the induced MT was revealed by immu-oelectron microscopic study (Fig. 8). Impact of iron overloading
s also reflected in the activity of aconitase enzyme, which plays aentral role in determining the labile iron pool and regulating thentracellular iron homeostasis. Any fluctuation in the labile ironool may result in impairment in the synthesis of iron-containing
ig. 7. FACS analysis of intracellular MT in p-DAB induced hepatocarcinogenesis. Hepatocyreatment and incubated with MT-primary antibody O/N. Non specific binding was blocACS Scan. MT was detected by a shift in green fluorescence (FL-1). Mean Fluorescence inontrol hepatocytes, (3) MT induction after 3 weeks of p-DAB treatment and (4) MT induc
Fig. 6. Antioxidant enzyme profile; (a) catalase activity expressed as rate of H2O2
decomposition at 240 nm. (b) SOD activity expressed as rate of NADH oxidation.*P = < 0.05, NS: not significant.
proteins or cell injury by prooxidants. Cytosolic aconitase is amb-ifunctional; serves as aconitase enzyme when iron levels are highand an as iron regulatory protein (IRP) regulating iron transportinto the cell during intracellular iron deficiency (Sreedhar and Nair,
2005). The observed decrease in aconitase activity in the 7-weektreatment group (Fig. 9) shows loss of its enzymatic activity dueto oxidative damage, providing further evidence in support of theincreased iron levels in the liver tissue.
tes isolated by two-step collagen perfusion method were permealised by methanolked by BSA. FITC labeled secondary antibody was then added and analyzed by BDtensity (MFI) was calculated by CellQuest software. (1) Unlabeled hepatocytes, (2)tion after 5 weeks of p-DAB treatment.
46 D. Mishra et al. / Toxicology Le
Fig. 8. Cytosolic localization of induced MT as revealed by immunoelectronmicroscopy.
F*
4
mcirichtroho
C
A
(
ig. 9. Aconitase activity of liver cytosol of mice treated with p-DAB for 7 weeks.P = < 0.001.
. Conclusions
The selective alteration in elemental profile in the p-DAB fedale mice indicates that such alterations are definite markers in the
arcinogenic initiation. More precise studies in this aspect defin-ng interaction of essential trace and ultratrace elements and theiratios in phases of carcinogenic induction might throw some lightn the direction of future research towards actual mechanism ofhemical carcinogenesis involving metabolic pathways and cellularomeostasis with respect to elemental concentration. We proposehat the preinitiation of liver neoplasia by treatment of p-DAB is theesult of oxidative damage secondary to the hemolytic depositionf iron in the liver. The study provides a plausible explanation ofow altered iron homeostasis can contribute to chemically inducedxidative stress and carcinogenesis.
onflicts of interest
There are no conflicts of interest.
cknowledgements
The author acknowledges the support of Institute of PhysicsIOP), Bhubaneswar for PIXE study and Dr. T. Biswas and Mr. A.
tters 203 (2011) 40–47
Biswas of National Institute of Cholera and enteric diseases (NICED),Kolkata for the FACS analysis.
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