Lipid Peroxidation Induced byExpandable Clay MineralsD A R I A K I B A N O V A , †

A N T O N I O N I E T O - C A M A C H O , ‡ A N DJ A V I E R A C E R V I N I - S I L V A * , § , ⊥ , ⊥

Facultad de Quımica, Universidad Nacional Autonoma de Mexico,Instituto de Quımica, Universidad Nacional Autonoma de Mexico,Circuito Exterior s/n, Ciudad Universitaria, Coyoacan, Mexico, DF04510, Mexico, Departamento de Procesos y Tecnologıa, Division deCiencias Naturales e Ingenierıa, Universidad AutonomaMetropolitana, Unidad Cuajimalpa (UAM-C), Artificios No. 40,6° Piso, C.P. 01120 Mexico, NASA Astrobiology Institute, and EarthSciences Division, Lawrence Berkeley National Laboratory,1 Cyclotron Rd., Berkeley, CA 94720

Received March 15, 2009. Revised manuscript receivedAugust 3, 2009. Accepted August 11, 2009.

Small-sized environmental particles such as 2:1 phyllosilicatesinduce oxidative stress, a primary indicator of cell damageand toxicity. Herein, potential hazards of clay particle uptakeare addressed. This paper reports that the content and distributionof structural Fe influence the ability of expandable clayminerals to induce lipid peroxidation (LP), a major indicator ofoxidative stress, in biological matrices. Three smectiteclays, hectorite (SHCa-1) and two nontronites (NAu-1) and (NAu-2) containing varying total content and coordination environmentof structural Fe, were selected. Screening and monitoringof LP was conducted using a thiobarbituric acid reactivesubstances (TBARS) assay. The higher content of TBARS innontronites than that in SHCa-1 suspensions was explainedbecause structural Fe contributes to LP. The observed lack ofcorrelation between TBARS content and the extent of Fedissolution indicated that the formation of TBARS is surfacecontrolled. Results showing a high TBARS content in SHCa-1 butnot in nontronite supernatant solutions was explainedbecause the former contains distinct, soluble chemicalcomponent(s) that could (i) induce LP by its (their) own rightand (ii) whose chemical affinity for organic ligands usedas inhibitors is weak. Clays serve as stronger inductors than 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) but aremuch weaker than FeSO4. The outcome of this work showsthat LP is clay surface-controlled and dependent on clay structuralcomposition.

IntroductionClays (d < 2 µm) are ubiquitous naturally occurring small-sized particles and are commonly found in a wide range ofenvironmental compartments, from sediments in the bottomof the ocean to atmospheric aerosols in the upper strato-

sphere. Although it has been recognized that the interactionof clays with organs may provoke health-deleterious con-sequences, to date there is little mechanistic understandingof the physiological effects due to exposure to clays. A recentstudy reports that long-term exposure of bare feet to alkalinesoils bearing high-clay contents has been related to non-infectious elephantiasis known as Podoconiosis (1). Colloid-sized silicate particles arguably enter through the skin causingendolymphangitis and obliteration of the lymphatic lumen(1). Another study on the bioavailability of hazardousmaterials to Wistar rats revealed that no macro-toxic effectswere observed if clays were administered orally (2). The samestudy, however, reports a positive relation between thecontents of trace-elements and clay accumulation in theorgans following the decreasing order of kidney > liver >heart > brain (2).

The biological activity of clays depends strongly onsuspension composition. For example, French green clayshave recently been shown to heal Buruli ulcer, a “flesh-eating”infection by Mycobacterium ulcerans (3). The authors of thatstudy concluded that (i) the chemistry of the water used tohydrate the clay poultices contained critical antibacterialagent(s), (ii) the chemistry was controlled by the clay mineralcomposition and surface properties, and (iii) the killingmechanism was not physical (attraction between clay andbacteria) but due to chemical transfer. A study on Ca-montmorillonite (SAz-2, Apache County, AZ) dissolution inthe presence of simulated extracellular lung (pH0 7.4) andlysosomal fluids (pH0 4.55) at 37 °C brought about siliconsteady state release values from 4.1 × 10-15 and up to 1.0 ×10-14 mol m-2 s-1, with incongruent, edge-controlled claydissolution (4). For a 1 µm grain, the measured range ofbiodurability was determined to range from 1300 to 3400yrs. Those results led to propose the potential use ofmontmorillonite to mitigate silica cytotoxicity (5).

Minerals can cause cell damage because of the productionof free radicals (6). Cell damage is commonly determined asthe progress of lipid peroxidation (LP). LP refers to theoxidative degradation of lipids in cell membranes. LP isinitiated by hydrogen abstraction from a methylene groupof a polyunsaturated fatty acid that forms a fatty acid radical.Subsequently, radicals undergo rearrangements and reactwith atmospheric triplet oxygen to produce lipid peroxideradicals, which can abstract more hydrogen. Finally, the chainreaction is terminated by reactions between radicals (7, 8).

In iron-environmental systems, redox chemistry under-pins LP. A study conducted in vitro in leukemic cells hadproposed that LP is initiated primarily by a Fe-O2 complexand to a lesser extent by Fenton and Haber-Weiss reactions(9). Another mechanistic study on LP in liposomes hasproposed that Fe2+ initiates LP on one hand and suppressesthe species responsible for the initiation on the other.Arguably, pre-existing lipid peroxides (LOOH) and lipidperoxyl radicals (LOO•) along with Fe2+ convey initiation (10).Cheng and Li (11) concluded that the redox chemistry ofiron plays an important role in the initiation of LP, andhydroxyl radicals can be dismissed as initiators. Iron oxo-species were found to be responsible for initial H abstraction(11). Finally, a recent work demonstrated that transitionmetals such as Fe(II), Cr(II), Pb(II), and Cd(II) generate LPin cod liver via Fenton-like reactions (12).

Clays constitute the most important of earth’s reservoirof iron, a key element for triggering fundamental metabolicpathways to sustain life. In this paper, the authors proposethat the content and distribution of structural Fe in expand-able clay minerals will influence the ability of clay minerals

* Corresponding author phone: (52) (55) 26 36 38 00 extension3827; fax: (52) (55) 26 36 38 00 extension 3832; e-mail: jcervini@igeograf.unam.mx.

† Facultad de Quımica, Universidad Nacional Autonoma de Mexico‡ Instituto de Quımica, Universidad Nacional Autonoma de

Mexico.§ Universidad Autonoma Metropolitana.⊥ NASA Astrobiology Institute.⊥ Lawrence Berkeley National Laboratory.

Environ. Sci. Technol. 2009, 43, 7550–7555

7550 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 19, 2009 10.1021/es9007917 CCC: $40.75 2009 American Chemical SocietyPublished on Web 08/21/2009

to induce LP in biological matrices. Three smectite clays,hectorite (SHCa-1) and two nontronites (NAu-1) and (NAu-2) bearing different total content and distribution of structuralFe {Fe(III)} were selected for this study (Table 1).

Herein, we report experimental determinations basedon thiobarbituric acid reactive substances (TBARS) forscreening and monitoring LP, a major indicator of oxidativestress. The assay provides important information regardingfree radical activity in disease states and has been used formeasuring antioxidant activity of several compounds andassessing LP. Of all organs in living systems, the brain istypically selected for conducting these assays because ofits high content of easily peroxidizable unsaturated fattyacids, high oxygen utilization accounting for one-fifth oftotal systemic consumption, and high content of Fe andascorbate (key ingredients in causing LP), and the brainis not rich in antioxidative enzymes (13). Experimentally,LP in brain homogenates can be induced in the presenceof Fe2+ (14, 15), 2,2′-azobis(2-amidinopropane) dihydro-chloride (AAPH) (16), H2O2 (17), or quinolinic acid (18, 19).The authors compare the effectiveness of expandable claysfor inducing LP with that of FeSO4 and AAPH.

Materials and MethodsSource of Clays. The <0.05 mm size fraction of nontronites(NAu-1, green color, Al-enriched; and NAu-2, brown color,Al-poor, contains tetrahedral Fe) from Uley Mine, SouthAustralia, and hectorite (SHCa-1, contains calcite) from San-Bernardino County, CA, were purchased from the SourceClays Repository of the Clay Minerals Society (PurdueUniversity, West Lafayette, IN).

Animals. Adult male Wistar rats (200-250 g) were providedby the Instituto de Fisiologıa Celular, Universidad NacionalAutonoma de Mexico (UNAM). Adult male Wistar rats wereapproved by the Animal Care and Use Committee (NOM-062-ZOO-1999). They were maintained at 25 °C on a 12/12h light-dark cycle with free access to food and water.

Rat Brain Homogenate Preparation. The animal sacrificewas carried out avoiding unnecessary pain. Rats weresacrificed with CO2. The cerebral tissue (whole brain) was

rapidly dissected and homogenized in phosphate bufferedsaline (PBS) solution (0.2 g of KCl, 0.2 g of KH2PO4, 8 g ofNaCl, and 2.16 g of NaHPO4 ·7 H2O/ L, pH adjusted to 7.4)as reported elsewhere (15, 20) to produce a 1/10 (w/v)homogenate. Then, the homogenate was centrifuged for 10min at 3400 rpm to yield a pellet that was discarded.

Protein Content in Brain Supernatant Solutions. Theprotein content in brain supernatant solutions was measuredusing the Folin and Ciocalteu’s phenol reagent (21) andadjusted to the desired concentration of either 2.86 or 3.33mg protein mL-1 with PBS solution.

Clay-Induced Lipid Peroxidation. All experiments wereconducted in an ice bath. Three hundred and fifty microlitersof the supernatant solution (2.86 mg protein mL-1) wereadded together with 50 µL of 10 µM EDTA to Eppendorftubes. The brain contains high levels of Fe that induce LP byitself (22). Adding EDTA to all samples served the purposeof chelating iron originally present in the brain homogenates.Then, 100 µL aliquots of clay suspensions in water were addedto Eppendorf tubes. The final concentration of protein andEDTA were 1 mg mL-1 and 1 µM, respectively. Then, themixture was incubated at 37 °C for 0.5, 1, 1.5, 2, 2.5, 3, 3.5,or 4 h in a Lab Line Titer Plate Shaker Model 4265 at a 1.5constant shaking speed.

For selected experiments it was necessary to adjust theamount of supernatant to about 300 µL in order to add 50µL of chelating agents [either EDTA or desferrioxamine Bmesylate salt (DFOB) from Sigma Chemical Co.]. For thispurpose, the initial protein content of the homogenate wasadjusted to 3.33 mg protein mL-1 to yield a final proteinconcentration in Eppendorf tubes of 1 mg mL-1.

Additionally, control experiments to test the induction ofLP were conducted in the presence of (i) brain only (no clay),(ii) FeSO4 (final concentration 10 µM), and (iii) AAPH (finalconcentration 20 mM), both obtained from Sigma ChemicalCo. (St. Louis, MO).

To discriminate the extent by which TBARS formation isassociated with soluble Fe versus structural Fe in claysuspensions, we conducted additional experiments. Claywater suspensions were centrifuged for 5 min at 12000 rpmto separate the supernatant solution and clay fraction.Experiments conducted in the presence of the supernatantsolution will be referred herein as SN-clay type. The clayfraction was resuspended in an equal water volume contentequivalent to the removed supernatant solution. Experimentsconducted in the presence of the resulting suspension willbe referred herein as resuspended clay (RS-clay type).

Thiobarbituric Acid Reactive Substances (TBARS) Quan-tification. A modified method described elsewhere (23) forTBARS quantification was used. The method is based on thereaction of malondialdehyde (MDA), a product of LP (24),and other TBARS (substances formed in the course of thereaction that reacting with TBA give an adduct whosespectrum is identical with that obtained from the MDAstandard (7)) with TBA in a 1:2 molar ratio on heating to givea red adduct whose concentration is related to the extent oflipid peroxidation (7, 8). A 1% (w/v) thiobarbituric acid (TBA)solution from ICN Biomedicalas, Inc. (Ohio) in 0.05 N NaOHwas prepared and mixed with 30% (w/v) trichloroacetic acidfrom Fluka (West St. Paul Milwaukee, MN) in 1:1 proportion.A one-half milliliter aliquot of the TBA reagent was added toeach Eppendorf tube. The tubes were cooled on ice for 10min, centrifuged at 3000 g (12000 rpm) for 5 min, and finallyheated at 95 °C for 30 min. The tubes were allowed to reachambient temperature. Two-hundred microliter aliquots ofthe supernatant solution were separated for analysis. Thecontent of TBARS in the solutions was determined by opticaldensity at λ ) 540 nm using a Bio-Tek EL × 808 microplatereader.

TABLE 1. Chemical Composition and Structural Formulae ofExpandable Clay Minerals

chemical composition (%)

hectorite SHCa-1a nontronite NAu-1b nontronite NAu-2b

SiO2 46.66 51.36 56.18Al2O3 0.86 8.150 3.114TiO2 0.04 0.201 0.020Fe2O3 0.32 35.94 37.85MnO - 0.013 0.016MgO 20.01 0.191 0.255CaO 14.01 3.565 2.342Na2O 1.35 0.033 0.143K2O 0.14 0.006 0.013P2O5 0.00 0.011 0.001SO3 - 0.001 0.004ZrO2 - 0.004 0.014Sr - 0.001 0.003

structural formulae

hectoriteSHCa-1c

(Na0.80) [Si7.90Al0.10][Mg5.30Li0.70]O20(OH)4

nontroniteNAu-1b

(Na1.05) [Si6.98 Al0.95Fe0.07][Al0.36 Fe3.61 Mg0.04]O20(OH)4

nontroniteNAu-2b

(Na0.72) [Si7.55 Al0.16Fe0.29][Al0.34 Fe3.54 Mg0.05]O20(OH)4

a Mermut and Cano, ref 26. b Gates et al., ref 27.c Schlegel et al., ref 28.

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The concentrations of TBARS were calculated by inter-polation from an experimental standard curve determinedfor tetrametoxipropane (TMP) as described elsewhere (25).The inhibition ratio (%) defined as the decrement of TBARSformation due to the effect of chelating agents was calculatedas follows

where C and E correspond to the absorbance for the controland test sample, respectively, at λ)540 nm. All data wererepresented as mean ( standard error of mean. One-wayANOVA followed by Dunnett’s test for comparisons againstcontrol were conducted for data analyses. Magnitude valueswith pe0.05 (*) and pe0.01 (**) were considered statisticallysignificant.

Determination of Dissolving Fe. After TBARS quantifica-tion, the supernatant of selected samples was separated inorder to carry out the determinations of total soluble Fe thatwere conducted by atomic absorption spectrometry using aVarian SpectrAA 110 instrument equipped with an acety-lene-air oxidant flame (AAS-F) at 248.3 nm.

Results and DiscussionIncubation Time and Clay Type and Concentration versusTBARS Production. Preliminary experiments were conductedto select an optimum incubation time (ti) so that all samplesachieved a significant LP level. Five-thousand ppm clay-watersuspensions were prepared. The final clay concentration inthe reaction vessels was 1000 ppm. Samples with ti ) 0 hwere used as reference (controls). The presence of clay hadan effect on TBARS formation (Figure 1). The type of clayinfluenced the extent of TBARS formation. For nontronites,TBARS formation with statistical significance (pe 0.01) wasdetected at tig 2 h. TBARS formation trends were similar forboth nontronites, with NAu-2 samples exhibiting a moreimportant effect on LP at tie 3 h. Samples bearing hectorite(SHCa-1), however, exhibited a more modest TBARS pro-duction, and statistical significance for TBARS formation wasdetected only for cases when ti g 2.5 h (Figure 1). Thus, ti )3 h was selected for conducting subsequent experiments.

Shown in Figure 2 are the results on the effect of the clayconcentration ({cc}) on TBARS formation. Samples containingnontronites exhibited similar trends in reactivity, conveyinga mild slope in the low {cc} region (< 200 ppm). By contrast,samples containing SHCa-1 showed an initial sharp slopefor TBARS formation, reaching maximum values about 3 nmolmg-1. Results shown in Figure 2 indicate that at lower clayloadings, SHCa-1 (0.32% {Fe2O3} (26)) exhibits activity that

is statistically equivalent to (if not greater than) other clayspossessing far greater Fe content (∼36% {Fe2O3} (27)), NAu-1, or NAu-2. Chemical analyses for SHCa-1 (Table 1) showthe presence of CaO and MgO in clay samples as received.The effect of CaO and MgO on TBARS formation has notbeen accounted for in the present study and deserves furtherscrutiny. We do not discard existing competing TBARSproduction mechanisms induced by clay surfaces and thesecomponents, however. This is further substantiated becausegiven effect of {cc} on TBARS production. Congruencybetween TBARS production and total Fe content (Table 1)holds true only for {cc}g 600 ppm. Likely, other mechanismsfor TBARS production in suspension may take place. Ad-ditional considerations on the role of clay surface area onTBARS production as affected by incubation time and clayconcentration are further discussed in the SupportingInformation.

Clay Surface versus Supernatant Solution Effectivenessas Lipid Peroxidation Inductor. Shown in Figures 3 areresults for TBARS formation as a function of the suspensionfraction, supernatant (SN), or resuspended (RS) clay. Sampleswith no clay were used as reference (control). Results forsamples containing RS NAu-1 or NAu-2 (Figure 3b,c) provide

FIGURE 1. Effect of incubation time on TBARS formation in (a)SHCa-1 (9), (b) NAu-1 (b), (c) NAu-2 (2) suspensions and brainhomogenate only (inverted triangles), with statisticalsignificance for p e 0.05 (*) and p e 0.01 (**) values.

Inhibition ratio (%) ) C - EC

× 100% (1)

FIGURE 2. Effect of clay concentrations on TBARS formation in(a) SHCa-1 (9), (b) NAu-1 (b), (c) NAu-2 (2) suspensions, withstatistical significance for p e 0.05 (*) and p e 0.01 (**) values.

FIGURE 3. Effectiveness of clay surface and supernatantsolution as TBARS formation inductors. (a) SHCa-1, (b) NAu-1,(c) NAu-2, with statistical significance for p e 0.05 (*) and p e0.01 (**) values.

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evidence to suggest that TBARS formation is coupled withthe presence of clay surfaces: RS fraction induced LP almostto the same extent than original clay suspensions. By contrast,TBARS contents in nontronite SN and control samples werecomparable. A different trend in reactivity was observed forthe case when the clay was SHCa-1. TBARS concentrationswere significantly higher (p < 0.01) in SHCa-1 than those incontrol samples. Unexpectedly, however, RS SHCa-1 sampleswere observed to contain higher TBARS concentrations (p< 0.05) than those on SHCa-1 suspensions before separationin the SN and RS fractions. Also, SN SHCa-1 samples werefound to have a significant activity toward inducing TBARSformation (p < 0.01).

To further elucidate whether TBARS formation is a claysurface-controlled reaction, we conducted additional experi-ments to underpin the participation of structural versusdissolved iron. Total amounts of supernatant iron after 3 hincubation time in 1000 ppm clay-water suspensions werequantified. The clay dissolution behavior varied with struc-tural formulas. Total dissolved Fe was detected to be as highas 7.1 ( 0.5 and 5.7 ( 0.6 ppm in NAu-2 and NAu-1 samples,respectively. By contrast, samples containing SHCa-1 showedtotal dissolved Fe to be as low as 0.7 ( 0.1 ppm, whichcompares to dissolved Fe amounts detected in the super-natant solution of samples containing no clay (brain ho-mogenate only), 0.3 ( 0.1 ppm. A comparison between thetotal Fe content in clays (Table 1) and TBARS production inthe presence of clay (Figure 3) shows a relation betweentotal Fe and TBARS formation. No relation became apparentbetween dissolved iron and TBARS production, however(Figure 3, SN fraction), suggesting the preferential participa-tion of structural over dissolving Fe to induce LP. However,the high TBARS production in SN SHCa-1 but not SNnontronite samples is consistent, again, with the idea thatdistinct soluble component(s) could trigger TBARS formation.

Surface areas-normalized curves after Figures 1 and 2(Figures 1S and 2S of the Supporting Information) indicatesimilarities between the extent of TBARS production inSHCa-1 and NAu-1 suspensions and a relatively higher TBARSproduction in NAu-2 suspensions. The variability in TBARSproduction by nontronites can be explained because ofexisting structural differences associated with distributionof structural iron over total structural iron content alone. Asdetermined by amplitude ratios 002 and 003 reflections ofCa-saturated films, deduced Fe3+ occupancy for NAu-1 andNAu-2 are reported to correspond to 0.26 and 0.39 (27),respectively. Reported X-ray pre-edge spectra for NAu-1 andNAu-2 samples reveal differences in intensity, position, andshape. Enhancements of the Fe K pre-edge intensity in NAu-2and the change in pre-edge profile (27) have been attributedto increased structural disorder due to lowering in symmetryaround 6-coordinated Fe3+ (29) or to appreciable amountsof Mg and/or Al within octahedral sites (30). Finally,experimental Fe3+-O waveforms for NAu-2 were found tobe lower in amplitude and shifted in comparison to NAu-1(27). Waveforms for NAu-2 are right-shifted at low Å-1. Suchphase shifts are consistent with shorter average Fe3+-O bonddistances and lower average coordination for NAu-2.

Inhibitors. Experiments were conducted to study theeffectiveness of organic ligands known to complex Fe (DFOB)and/or other metals (EDTA) for inhibiting TBARS formation.Results are shown in Figure 4. The effectiveness of the ligandfor inhibiting TBARS formation depended on the (a) ligandmolecular structure and initial concentration and (b) pres-ence and type of clay. The effectiveness of DFOB for inhibitingTBARS production was found to be higher than 60% for caseswhen DFOB initial concentration surpassed 2 µM, irrespectiveof the clay structural formulae. EDTA was observed to be aneffective inhibitor of TBARS formation in nontronite suspen-sions. In suspensions bearing SHCa-1, DFOB resulted to be

a more effective inhibitor than EDTA. So is the case forsuspensions bearing NAu-1, yet differences in ligand ef-fectiveness were found to be less pronounced. However, theeffectiveness of DFOB and EDTA in NAu-2 suspensions wascomparable, irrespective of initial ligand concentration.Different trends in panels b and c of Figure 4 can be explainedbecause NAu-2 has a slightly higher iron content (Table 1).Assuming the participation of structural Fe in LP, it followsthat a larger concentration of DFOB and EDTA would beneeded than in the case of NAu-1 in order to achievecomparable inhibition activity levels.

The amount of {Fe2O3} in SHCa-1 is small (0.32%) (26)compared to those reported for NAu-1 (35.21%) or NAu-2(37.85%) (27). However, at low ligands concentrations lesssignificant inhibition was achieved than in the case ofnontronites. These results are explained because SHCa-1suspensions contain distinct, soluble chemical component(s)(Table 1) that could (i) induce LP by its (their) own right and(ii) whose chemical affinity for DFOB or EDTA is weak.

Experiments to study the % inhibition of TBARS formationin the presence of EDTA but not DFOB are consistent withthe idea of ligand specificity and that metal centers otherthan Fe may also contribute to TBARS formation. A shallowerslope for TBARS formation in SHCa-1 suspensions (Figure2) is explained because clay surfaces may be competing withother processes triggering TBARS formation in suspension.Still, a higher TBARS concentration in nontronites samplesprovides evidence to support the hypothesis that structuralFe contributes to LP.

Lipid Peroxidation Inductors. Listed in Table 2 are resultspertinent to the relative effectiveness of expandable clays asLP inductors. For the purpose of comparison, the peroxi-dation extent by clays (1000 ppm), AAPH (20 mM), and FeSO4

(10 µM) was normalized to ppm per inductor. FeSO4 wasselected as a reference reagent because it has been reportedas an excellent oxidative stress inductor (18). AAPH is a water-

FIGURE 4. Effect of organic ligands in inhibiting TBARSformation. Solid circles ) DFOB, open circles ) EDTA. (a)SHCa-1, (b) NAu-1, and (c) NAu-2.

TABLE 2. Activity of Expandable Clay Minerals, AAPH, andFeSO4 as Initiators of Lipid Peroxidation

inductorTBARS/prot.(nmol mg-1)

TBARS/prot.(nmol mg-1 ppm-1)

SHCa-1 (1000 ppm) 2.49 ( 0.24 2.49 × 10-3

NAu-1 (1000 ppm) 5.83 ( 0.42 5.83 × 10-3

NAu-2 (1000 ppm) 6.68 ( 0.41 6.68 × 10-3

AAPH (20 mM) 3.36 ( 0.78 6.20 × 10-4

FeSO4 (10 µM) 10.28 ( 0.47 3.70 × 10

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soluble azo compound that is used extensively as a free radicalgenerator. AAPH decomposition produces molecular nitro-gen and two carbon radicals. The carbon radicals may thencombine to produce stable products or react with molecularoxygen to yield peroxyl radicals prior inducing LP (31). Onthe one hand, AAPH and Fe2+ are hydrophilic and water-soluble (32), whereas clays form water suspensions. Theinitiator AAPH produce radicals at a constant rate (33), leadingto the formation of hydroperoxide radical. On the other hand,in the presence of oxygen, Fe2+ initiates the production ofOH• radicals. Also, soluble iron(II) could react with lipidperoxides to form alkoxyl radicals at 1.5 × 103 M-1 s-1 (34).It is well-known that Fe2+ acts as the most efficient LPinductor. Yet, it does not become clear if this is because ofthe type of radical produced or the quantity of radicalsgenerated during the exposure time (32). In this context, ourresults show that clays served as stronger inductors thanAAPH but were much weaker than FeSO4. We have explainedour results because clays contain structural iron, predomi-nantly in the form of structural iron(III) [neither nontronitecontains measurable iron(II) (35)]. Hence, our hypothesis isthat LP initiation by clays resembles that proposed involvingthe formation of perferryl ion through Fe3+/O2

•-, whereas LPinitiation by FeSO4 resembles that by Fe2+/O2 (9). Given thatcell superoxide concentrations are much lower over oxygenconcentrations (36), it follows that LP induced by clays occursslower compared to LP induced by FeSO4.

Environmental RelevanceThis manuscript provides evidence to show that small-sizedenvironmental particles such as 2:1 phyllosilicates can induceoxidative stress via LP. The manuscript presents data tosupport that LP is surface-controlled and largely dependenton structural composition. As determined by TBARS forma-tion, the structural iron in clays was found to be importantfor the ability of expandable clays to induce LP. The outcomeof this work contributes to further understanding of howsmall-sized particles induce changes in metabolic toxicitypathway(s).

LP induced by Fe-rich expandable clay minerals (∼36%{Fe2O3} (27)), is coupled with the presence of clay surfacesand is affected by the distribution of structural Fe (Figures1 and 2, 3b,c and Figures 1S and 2S of the SupportingInformation). A different trend in reactivity in Fe-poor (SHCa-1) clay suspensions (0.32% {Fe2O3}) (26) was observed,however. Supernatant SHCa-1 samples were found to havea significant activity toward inducing TBARS formation(Figure 3a), in consistency with the idea that the presenceof distinct, soluble chemical component(s) that could induceLP by its (their) own right. These findings expand on a report(3) concluding that bactericidal activity of two French clays,namely, CsArO2 and CsAgO2 (with similar total Fe content,about 6% Fe2O3, 115.4 m2 g-1), relates to the presence ofsoluble components including Cs, As, Se, Cd.

Exposure of biological material to small-sized environ-mental particles has been related to a variety of healthproblems. Air pollution particles reportedly catalyze thegeneration of free-radical reactions coupled with lung damage(37). The interaction of dust particles with alveolar mac-rophages can induce LP (38). As evidenced by electronmicroscopy of animal and human tissue, respirable claymineral dusts results in muscovite deposition (39). Inhalationand retention of dust particles formed from certain mineralspecies such as zeolites and fibrous amphiboles has beenrelated to lung cancer (40-42). However, prolonged exposureof rats to high concentrations of kaolinite (300 mg m-3) hasbeen reported as lethal (43).

How does the association of small-sized particles withbiomolecules induce changes in toxicity? The answer to this

question is still unknown and deserves scrutiny. In this regard,little is known about interactions of long-chain biologicalmolecules at interfaces of natural small-sized particles,thereby there is a lack of understanding of processes suchas proton exchange, metal complexation, or electron transfer.Also, the chemical behavior is largely determined by theirinteractions with water. The characterization of the natureof clay-water interactions with biomolecular-structuralsubunits at the molecular level becomes necessary to gaina better understanding of toxicity mechanisms. Becausebiomolecules influence electron transfer processes that occurin natural particles, their surface complexation is thought toregulate metal toxicity, similar to redox-active metal ions insolution (44, 45).

AcknowledgmentsThe authors express gratitude to M. Sc. Pilar FernandezLomelin [Instituto de Geografia, Universidad Nacional Au-tonoma de Mexico, (UNAM)] for technical support and Dr.Hugo Destaillats (Lawrence Berkeley National Laboratory)for helpful comments. D.K. is thankful for the support of aDGAPA-UNAM undergraduate scholarship. This project wassupported in part by UNAM (PUNTA-PAPIIT Grant IN116007-2), CONACYT (SEP-CONACYT Ciencia Basica 2006, Grant61670), and by ECACORE 2020 (SEMARNAT-CONACYT).

Note Added after ASAP PublicationThis paper was published ASAP on August 21, 2009 with anerror in Figure 1 and other typographical errors. The correctedversion was published ASAP on August 31, 2009.

Supporting Information AvailableAdditional considerations on the effect of the clay type andsurface area on TBARS production, and figures normalizedto clay specific surface area. This information is availablefree of charge via the Internet at http://pubs.acs.org.

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