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Chemosphere 82 (2011) 418423

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Chemosphere
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Determination of lipid peroxidation and cytotoxicity in calcium, magnesium, titanium and hectorite (SHCa-1) suspensions
Daria Kibanova a, Antonio Nieto-Camacho b, Teresa Ramrez-Apan b, Javiera Cervini-Silva c,d,e,
a

Facultad de Qumica, Universidad Nacional Autnoma de Mxico, Mexico Instituto de Qumica, Universidad Nacional Autnoma de Mxico, Mexico c Universidad Autnoma Metropolitana, Unidad Cuajimalpa, Mexico d NASA Astrobiology Institute, United States e Earth Sciences Division, Lawrence Berkeley National Laboratory, United States
b

a r t i c l e

i n f o

a b s t r a c t
This paper reports data on the relative ability of CaO, CaCl2, MgO, MgCl2, TiO2, and hectorite (SHCa-1) to induce oxidative stress (as determined by lipid peroxidation, LP) in biological matrices. The effectiveness of structural (oxide form) versus soluble Ca and Mg to induce LP is compared. An assessment on cytotoxicity as affected by soluble and structural Ca, Mg, TiO2 and SHCa-1 is also addressed. LP was screened and monitored using the Thiobarbituric Acid Reactive Substances (TBARS). The extent of TBARS production was found to vary with the type and initial concentration of the soluble or structural cation, Ca or Mg respectively. Obtained results showed higher magnitude values for the latter set of experiments. In the presence of TiO2 no signicant TBARS production was detected pointing out a negligible effect of TiO2 on LP. At solid concentrations ca. 100 ppm, CaO appears to be more effective than SHCa-1 to induce LP. By contrast at ca. 25 ppm, MgO appears to be more effective than the clay mineral. The SHCa-1 LPinducing activity has been proven to closely relate to structural Ca. The prevalence of mechanisms that may induce LP but not cytotoxicity (as determined by cell growth inhibition) was also addressed. Results on cell growth inhibition as affected by soluble and structural Ca, Mg, TiO2 and hectorite provide evidence to support that structural Ca or Mg brings about signicantly higher variations than soluble Ca. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 28 April 2010 Received in revised form 20 September 2010 Accepted 26 September 2010 Available online 20 October 2010 Keywords: Lipid peroxidation Small-sized mineral toxicity Calcium Magnesium Hectorite

1. Introduction Lipid peroxidation (LP) refers to the oxidative degradation of lipids in cell membranes resulting in cell damage. LP is initiated by hydrogen abstraction from a methylene group of a polyunsaturated fatty acid that forms a fatty acid radical. Subsequently, radicals undergo rearrangements and react with atmospheric triplet oxygen to produce lipid peroxide radicals, which can abstract more hydrogen. Finally the chain reaction is terminated by reactions between radicals (Fernndez et al., 1997; Wheatley, 2000). The measurement of Thiobarbituric Acid Reactive Substances (TBARS) (Ohkawa et al., 1979) has become the method of choice for screening and monitoring LP, a major indicator of oxidative stress. The assay provides important information regarding free radical activity in disease states and has been used for measuring

Corresponding author. Address: Departamento de Procesos y Tecnologa, Divisin de Ciencias Naturales e Ingeniera, Universidad Autnoma Metropolitana, Unidad Cuajimalpa (UAM-C), Articios No. 40, 6 Piso, Col. Miguel Hidalgo, Delegacin lvaro Obregn, C.P. 01120 Mxico, D.F., Mexico. Tel.: +52 55 26 36 38 00x3827; fax: +52 55 26 36 38 00x3832. E-mail address: jcervinisilva@yahoo.com (J. Cervini-Silva).
0045-6535/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.09.069

anti-oxidant activity of several compounds and assessing LP. Of all organs in living systems, the brain is typically selected for conducting assays to measure oxidative stress. This is because the brain has (1) a high content of easily-peroxidizable unsaturated fatty acids, (2) a high oxygen utilization accounting for one-fth of total systemic consumption, (3) a high content of both Fe and ascorbate (they are key ingredients in causing LP), and that (4) it is not rich in antioxidative enzymes (Floyd and Hensley, 2002). Experimentally, LP in brain homogenates can be induced in the presence of Fe2+, AAPH, H2O2, or quinolinic acid (Rios and Santamaria, 1991; Jara-Prado et al., 2003). The effect of minerals on oxidative stress as determined by LP has been recently elucidated (Kibanova et al., 2009). In particular, the <0.05 mm size fraction of nontronites (NAu-1 and NAu-2) from Uley Mine South Australia and hectorite (SHCa-1) from San Bernardino County, California, USA were subject of study. Physicochemical properties associated with clay structural Fe were found to affect LP (as determined by TBARS production) in Fe-rich ($36% Fe2O3) nontronite suspensions. Specically, LP was observed to be clay surface-controlled, and affected by the content and distribution of structural Fe. A different trend in reactivity in Fe-poor, hectorite suspensions (Fe2O3% 60.32) was recorded, however. High

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levels of LP observed in SHCa-1 but not in nontronite (NAu-1 or NAu-2) supernatant solutions was interpreted to mean that the presence of distinct soluble component(s) triggers LP (Kibanova et al., 2009). An inference made based on reported chemical analyses for SHCa-1 (Mermut and Cano, 2001) pointed to the plausible presence of CaO, MgO and TiO2 in clay samples. However, the effect of small-sized CaO, MgO and TiO2 on LP was not accounted for by and it is subject to study in the present work. Calcium-induced LP in mammals (Braughler, 1988; Mirabelli et al., 1989; Pascoe and Reed, 1989) or plants (Dhindsa et al., 1982; Leshem et al., 1984) has received little attention. Stimulation of LP by low Ca2+ concentrations has been observed under a variety of conditions (Jain and Shohet, 1981; Beatrice et al., 1984; Perry and Epel, 1985; Richter and Frei, 1985; Malis and Bonventre, 1986; Braughler, 1988; Mirabelli et al., 1989). Inhibition of LP by high calcium concentrations has only been observed at Ca2+ levels above 1 mM in liposomes (Babizhaev, 1988) and in heart mitochondria and microsomes (Kagan et al., 1983), which has little relevance to physiological conditions, i.e. concentrations below 10 lM. A dose-normalized pulse-radiolysis study shows that the production of O at pH 10.4 in the absence of Ca2+ and with Ca2+ 2 concentrations of 0.051 mM were identical within experimental error (Bors et al., 1990). As determined by the Electron Paramagnetic Resonance (EPR) spectrum of the superoxide radical species, O (Bray et al., 1976), the presence of 0.3 mM Ca2+ caused elec2 tronic alterations; yet, no conclusion on complex formation were provided. Those results are in agreement with reports on the lack of either spectroscopic or kinetic evidence for the formation of CaO complex(es) at Ca2+ concentrations ca. 1 mM (Yagi and Miyazaki, 1988). Such Ca2+ concentrations correspond to maximal physiological levels in animal systems. The extent of LP and alterations in collagen metabolism in the hearts in rats fed a magnesium-decient diet for 28, 60, or 80 d has been reported (Kumar et al., 1997). Reportedly, after 60 d of treatment, LP (as determined by TBARS production) at the cardiac tissue level was observed to increase by 39%. Arguably, the extent of LP is affected by dietary deciency in magnesium. Results provided in that study also reveal a net deposition of collagen coupled with alterations in the Mg tissue levels. Another study provides data to show that Wistar rats fed with a severe magnesiumdecient diet brought about mineral homeostasis, increased LP, and reduced Mg2+/Ca2+ antagonism (Vormann et al., 1995). Other study focused on testing the effect of low extracellular magnesium ions, [Mg2+]0 (0 6 [Mg2+]0 6 0.48 mM) on LP tested in primary cultured cerebral vascular smooth muscle cells (Altura et al., 2003) revealed concentration-dependent rises in malonaldehyde (MDA) after 3 h of exposure to low [Mg2+]0 rising to levels 312 normal after 1824 h. In that study, MDA levels were found to be inversely proportional to [Mg2+]0, providing evidence to suggest that low [Mg2+]0 levels induce LP. In this paper the authors compare the ability of CaO, CaCl2, MgO, MgCl2, TiO2, and hectorite (SHCa-1) to induce LP in biological matrices. The authors also compare the effectiveness of soluble (CaCl2, MgCl2) versus structural (CaO, MgO) forms to induce LP. Herein, we report experimental results based on TBARS determination. Finally, the authors assess% cell growth inhibition (cytotoxicity) as affected by soluble and structural Ca, Mg, TiO2, and SHCa-1.

lowing chemical composition (Mermut and Cano, 2001): 46.66% SiO2, 0.86% Al2O3, 0.04% TiO2, 0.32% Fe2O3, 20.01% MgO, 14.01% CaO, 1.35% Na2O, 0.14% K2O. CaO, MgO, CaCl2 and MgCl2 were purchased from Sigma Aldrich and TiO2 from Evonik (Evonik Degussa P25). 2.2. Animals Adult male Wistar rats (200250 g) were provided by the Instituto de Fisiologa Celular, UNAM. Adult male Wistar rats were approved by the Animal Care and Use Committee (NOM-062ZOO-1999). They were maintained at 25 C on a 12/12 h lightdark cycle with free access to food and water. 2.3. Rat brain homogenate preparation The animal sacrice was carried out avoiding unnecessary pain. Rats were sacriced with CO2. The cerebral tissue (whole brain) was rapidly dissected and homogenized in phosphate buffered saline (PBS) solution (0.2 g KCl, 0.2 g KH2PO4, 8 g NaCl and 2.16 g NaHPO47H2O L1, pH adjusted to 7.4), as reported elsewhere (Rossato et al., 2002; Domnguez et al., 2005) to produce a 1/10 (w/v) homogenate. Then, the homogenate was centrifuged for 10 min at 1100g (ca. 3400 r.p.m.) to yield a pellet that was discarded. 2.4. Protein content in brain supernatant solutions The protein content in brain supernatant solutions was measured using the Folin and Ciocalteus phenol reagent (Lowry et al., 1951) and adjusted to 2.86 mg protein mL1 with PBS solution. 2.5. Induced lipid peroxidation All experiments were conducted in ice bath. Three hundred and fty microliters of the supernatant solution (2.86 mg protein mL1) were added together with 50 lL of 10 lM EDTA to Eppendorf tubes. The brain contains high levels of Fe that induce LP by its own right (Floyd, 1999). Adding EDTA to all samples served the purpose of chelating iron originally present in the brain homogenates. Then, 100 lL aliquots of solid suspensions or solutions in water were added to Eppendorf tubes. Final concentrations of test compounds are reported in Table 1 and Fig. 1. Final concentration of protein and EDTA were 1 mg mL1 and 1 lM, respectively. Then, the mixture was incubated at 37 C for 3 h in a Lab Line Titer Plate Shaker Model 4265 at 1.5 constant shaking speed. 2.6. Thiobarbituric Acid Reactive Substances (TBARS) quantication A modied method described elsewhere (Ohkawa et al., 1979) for TBARS quantication was used. The method is based on the reaction of malondialdehyde (MDA), a product of LP (Dotan et al., 2004), and other TBARS (substances formed in the course of the reaction that reacting with TBA give an adduct whose spectrum is identical with that obtained from MDA standard (Fernndez et al., 1997)) with TBA in a 1:2 M ratio on heating to give a red adduct whose concentration is related to the extent of LP (Fernndez et al., 1997; Wheatley, 2000). A 1% (w/v) TBA solution in 0.05 N NaOH was prepared and mixed with 30% (w/v) TCA in 1:1 proportion. A half-mL aliquot of the TBA reagent was added to each Eppendorf tube. The tubes were cooled on ice for 10 min, centrifuged at 3000g (ca. 12 000 r.p.m.) for 5 min, and nally heated at 95 C for 30 min. The tubes were allowed to reach ambient temperature. Two-hundred microliters aliquots of the supernatant solution were separated for analysis. The content of TBARS in the

2. Materials and methods 2.1. Test compounds Hectorite (SHCa-1) from San Bernardino County, CA was purchased from the Source Clays Repository of the Clay Minerals Society (Purdue University, West Lafayette, IN). SHCa-1 has the fol-

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Table 1 TBARS production ({TBARS}) in CaO, MgO, TiO2, and SHCa-1 suspensionsa,b. CaO [CaO] (ppm) 1.1 3.6 11.2 35.4 112 353.9
a b c

MgO {TBARS}Ca 0.64 0.09 0.83 0.08 1.58 0.10 1.89 0.05 3.95 0.10a 0.59 0.03 [MgO] (ppm) 0.8 2.6 8.1 25.5 80.6 255 {TBARS}Mg 0.63 0.05 0.72 0.04 0.76 0.03 2.70 0.04a 0.52 0.03 0.45 0.04

TiO2 [TiO2] (ppm) 1.6 5.1 15.9 50.5 160 505 {TBARS}Ti 0.51 0.07 0.47 0.07 0.49 0.06 0.43 0.06 0.32 0.09 0.31 0.12

SHCa-1c [SHCa-1] (ppm) 100 177.8 316.2 562.4 1000 {TBARS}SHCa-1 1.17 0.33 1.52 0.26 2.65 0.22 3.20 0.21a 2.73 0.27

Bold magnitude values denote maximum TBARS production detectable. Units for TBARS production are nmol TBARS mg protein1. Data taken from Figure 2 in Kibanova et al. (2009).

1 .6

4.5
CaCl2 MgCl2

nmol T BARS / mg protein

**

nmol T BARS / mg protein

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.01

**

4.0 3.5 3.0 2 .5 2.0 1.5 1.0 0.5 0 .0 0.01

CaO MgO TiO 2

**

** **

** *

0.1

10

0.1

10

Concentration (mM)
4.5
CaO CaCl C Cl2

Concentration (mM)
3.0
MgO MgCl M Cl2

nmol T BAR S/ mg prote in

4.0 3.5 35 3.0 2.5 2.0 1.5 1.0 0.5 5 0.0 0.01

**

C
nmol T BARS / mg protein

**

2.5 2.0 1.5 15 1.0 0.5 0.0 0.01

** * *
0.1

**

**

**

10

0.1

10

Concentration (mM)

Concentration (mM)

Fig. 1. Effect of concentration on the production of TBARS in the presence of: (A) soluble Ca (open circles) and Mg (open triangles); (B) structural Ca (bold circles), Mg (bold triangles), or Ti (bold squares); (C) structural (bold circles) and soluble (open circles) Ca; (D) structural (bold triangles) and soluble (open triangles) Mg. Statistical signicance determined experimentally corresponded to p 6 0.05() and p 6 0.01() values.

suspensions was determined by optical density at k = 540 nm using a Bio-Tek EL 808 Microplate Reader. The concentrations of TBARS were calculated by interpolation from an experimental standard curve determined for tetrametoxipropane (TMP) as described elsewhere (Esterbauer and Cheeseman, 1990). All data were represented as mean standard error. One-way ANOVA followed by Dunnetts test for comparisons against control were conducted for data analyses. Magnitude values with p 6 0.05() and p 6 0.01() were considered statistically signicant.

2.7. Cytotoxicity assay The cytotoxic effect was determined on human lymphocytes MT 2 cells, using the protein-binding dye sulforhodamine B in microculture assay to measure cell growth inhibitory activity (Monks et al., 1991). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 000 units mL1 penicillin G sodium, 10 000 lg mL1 streptomycin sulfate and 25 lg mL1 amphotericin B (Gibco). The cultures were maintained at 37 C in 95:5 H2O:CO2 atmosphere.

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For the assay, cells were removed from the tissue culture asks and diluted with fresh media. One-hundred microliter cell suspension aliquots were added to the wells of a 96 well microtiter plates (Costar). The concentration was 5 104 cell per well. The suspensions were then incubated for 4 h at 37 C. Stock solutions of test compounds in water were prepared and diluted. One-hundred microliter aliquots were added to the wells and the nal test compounds concentration was adjusted to 100 lM (or 100 ppm for SHCa-1). The resulting suspensions were incubated for 24 h at 37 C, then the cultured cells were xed in situ by adding 50 lL of cold 50% (w/v) TCA. Once again, the culture cells were incubated for 1 h at 4 C. Next, the supernatant solutions were discarded and the plates were washed three times with water, and then air-dried. The obtained cultured cells xed with TCA were stained for 30 min with 100 lL of 0.4% SRB solution. The resulting protein-bound dye was extracted with 10 mM unbuffered Tris base. The plates were placed on a shaker for 5 min prior to UV analyses. Optical densities were determined in an Ultra Microplate Reader (Elx 808, BIO-TEK Instruments, Inc.) at k = 515 nm. The cell growth inhibition was calculated according to:

whereas that corresponding to soluble Ca ({TBARS}Ca2+) was found to decrease after 1 mM, showing a narrower activity range. In contrast at concentrations surpassing 6 mM, the presence of either source of Ca resulted in the cessation of TBARS production. Fig. 1D compares {TBARS}Mg and {TBARS}Mg2+ magnitude values. For the case when the initial concentration of magnesium was 60.2 mM, either as MgO or Mg2+, trends for the production of TBARS, {TBARS}Mg and {TBARS}Mg2+, were found to be comparable. However, for the case when MgO was the source of Mg and 0.63 mM was the initial concentration, signicant increases in activity were detected. That was not the case for when Mg2+ was the source of Mg, with no signicant trend changes observed over the entire concentration range tested. Magnesium was found to present a narrow activity region and only when in the structural form. 3.2. Role of structural calcium and magnesium on TBARS production by SHCa-1 Listed in Table 1 are {TBARS} values detected in SHCa-1 bearing suspensions (data taken from Kibanova et al. (2009)) {TBARS}SHCa-1, and {TBARS}Ca, {TBARS}Mg, {TBARS}Ti. As evidenced by {TBARS}Ca and {TBARS}SHCa-1 values, at solid concentrations ca. 100 ppm, CaO appears to be more effective than SHCa-1, to induce LP. On the other hand, at solid concentrations as low as ca. 25 ppm, MgO appears to be nearly as effective as the clay mineral present at higher concentrations (ca. 3001000 ppm) to induce LP. LP-inducing activity of SHCa-1 and structural Ca and Mg was further compared. Considering the clay composition, CaO or MgO content was calculated for each of clays concentrations as follows:

% inhibition 100

  ODcellstestcompound ODblank

where ODblank is the optical density of cells only. Analyses were conducted by quatruplicates. 3. Results and discussion 3.1. Ca, Mg and Ti induced LP Fig. 1 illustrates the effect of structural and soluble Ca and Mg and TiO2 on LP (as quantied by TBARS production, {TBARS}). For the case of soluble compounds, the extent of {TBARS} varied with the type and initial concentration of the cation, namely [Ca2+]0 or [Mg2+]0. The production of TBARS was named {TBARS}Ca2+ or {TBARS}Mg2+, correspondingly (Fig. 1A). Soluble calcium, Ca2+, presented a marked effect on {TBARS} depending on concentration, denoting a maximum TBARS formation at 0.63 mM concentration. Soluble magnesium, Mg2+, however, showed a much weaker activity towards {TBARS}, with a mild slope in the concentrations range tested. Magnitude values for {TBARS}Ca2+ were found to surpass those for {TBARS}Mg2+ for the cases when [Ca2+]0 and [Mg2+]0 6 1 mM. On the other hand, the effectiveness of Ca2+ on TBARS production decreased markedly for the cases when the initial concentration surpassed 0.63 mM and, noteworthingly, {TBARS}Ca2+ magnitude values were found to fall even below {TBARS}Mg2+ magnitude values. Fig. 1B presents the activity of the oxides tested towards inducing LP, namely CaO, MgO and TiO2 ({TBARS}Ca, {TBARS}Mg, {TBARS}Ti). Like for the case of {TBARS}Ca2+ and {TBARS}Mg2+, {TBARS} magnitude values were found to depend on the type of structural cation. CaO and MgO showed activity on {TBARS}, however MgO activity range resulted to be narrower than for CaO. On the other hand, {TBARS}Ti values were found not to depend on solid concentration, and approximated 60.5 nmol TBARS mg1 protein showing a negligible effect of TiO2 on {TBARS}. A direct comparison of the effect of CaO and Ca2+ and MgO and Mg2+ on LP as function of the initial concentration is considered next. Fig. 1C shows the effect of Ca initial concentration on the production of TBARS in suspensions containing structural or soluble Ca, {TBARS}Ca or {TBARS}Ca2+, respectively. Values for {TBARS}Ca and {TBARS}Ca2+ were found to be comparable for when Ca initial concentration 61 mM. The effectiveness of structural Ca to induce TBARS production ({TBARS}Ca) was found to increase markedly,

CaOSHCa-1 0:1401 SHCa-1 MgOSHCa-1 0:2001 SHCa-1

2 3

where the values 0.1401 and 0.2001 represent the fraction of calcium or magnesium oxide in the clay, respectively (Mermut and Cano, 2001). Results are presented in Fig. 2. Fig. 2A compares the effectiveness of CaO as a free oxide versus CaO contained in hectorite, CaOSHCa-1. Both trends are similar, conveying pronounced slopes at the low concentration region, passing through a maximum and the subsequent decaying of the activity. Structural Mg results are listed in Fig. 2B. Contrasting to the previous case, in spite of having rising-and-falling trends, data graphs do not superimpose. MgO activity completely decays at 80.6 ppm, while in the case of MgOSHCa-1 it keeps rising until [MgO]SHCa-1 = 112 ppm and then slowly diminishes. It becomes clear at this point that in spite of high MgO content in SHCa-1, {TBARS}SHCa-1 is more consistent with {TBARS}Ca over {TBARS}Mg magnitude values. 3.3. Cell growth inhibition induced by soluble and structural Ca, Mg, Ti and SHCa-1 Data shown in Figs. 1 and 2 and Table 1 provide evidence to show that, as determined by the production of TBARS, the presence of soluble or structural calcium or magnesium induces lipid peroxidation. These ndings were found to oppose results pertinent to cytotoxicity. The presence of soluble or structural Ca, Mg, or Ti, or SHCa-1 in suspension had little effect on cell growth inhibition. Cell growth inhibition was found to vary as function of the chemical composition according to: CaO (17 1.2%) > MgCl2 (12.7 1.2%) $ MgO (12.6 0.7%) > SHCa-1 (9.9 1.2%) $ TiO2 (9.2 0.4%) > CaCl2 (5.8 1.2%) (Fig. 3). Our results can be summarized by noting that the type of cation has an effect on both, TBARS production and cell growth inhibition behaviors. First, {TBARS} in suspensions bearing high

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4.5 4.0

CaO CaOSHCa.1

3.5

MgO MgOSHCa-1

nmol TBARS / mg protein

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 50 100 150 200 250 300 350

nmol TBARS / mg protein

3.0 2.5 2.0 1.5 10 0.5 0.0 0 50 100 150 200 250

CaO (ppm)

MgO (ppm)

Fig. 2. Effect of structural Ca and Mg on the production of TBARS by SHCa-1.

20 18 16 14

12 10 8 6 4 2 0 CaO MgO TiO2 CaCl2 MgCl2 SHCa-1

Fig. 3. Cytotoxic effect on human lymphocytes MT 2 cells (% cell growth inhibition).

concentrations of CaO (Fig. 1) surpassed {TBARS} in suspensions bearing any other source of Ca, Mg, Ti, or SHCa-1. Likewise, cytotoxicity determinations reveal that suspensions of CaO show cell growth inhibition behavior which surpasses that proper of suspensions bearing any other tested compounds. It is worth noting that the presence of CaO resulted in a cell growth inhibition behavior, which was as much as three times that measured in suspensions bearing Ca2+. Second, Mg suspensions bearing low Mg initial concentrations showed similarities in {TBARS} in the presence of soluble or structural Mg. Cell growth inhibition measurements provide an indication that magnitude values at 100 lM concentration are comparable, whether the source of Mg is in the structural or soluble form. Third, TiO2 led to 9.2% cell growth inhibition, yet no LP as determined by {TBARS}, was recorded (Fig. 1). A previous report (Mabile et al., 1995) provides evidence to show a lack of correlation between cytotoxicity and {TBARS}. On the other hand, our results coincide with reports on TiO2 nanoparticles induced cytotoxicity (Warheit et al., 2006; Jin et al., 2008), and they are congruent with studies proving that TiO2 fails to induce statistically signicant TBARS formation in lung homogenates (Olmedo et al., 2008). 4. Conclusions Based on the results presented in this manuscript, the following conjectures are proposed:

Results in Fig. 1 indicating low {TBARS} in the presence of Ca2+ at concentrations above 0.6 mM agree well with the outcome of a study conducted on liposomes (Babizhaev, 1988), heart mitochondria, and microsomes (Kagan et al., 1983) to study the effect of calcium on LP. In that study, 1 mM Ca2+ concentrations were observed to bring about the inhibition of LP. The observed positive relation, albeit smooth, between {TBARS} and Mg2+ concentrations in suspension, on the other hand, contrast to studies that report that a magnesium-decient diet causes LP in rats (Vormann et al., 1995; Kumar et al., 1997). Cytotoxicity experiments determined in lymphocytes MT 2 cells reveal a higher cell growth inhibition in suspensions bearing 100 lM Mg2+ over Ca2+. Given that Ca and Mg own distinct biological functions, the present results on cell growth inhibition are thought to be due to the prevalence of metabolic pathways related to LP but not necessarily to cytotoxicity, and that can be mutually exclusive (Dogterom et al., 1989). Structural Ca brings about significantly higher biological variations than soluble Ca as determined by cell growth inhibition. These results coincide with results on TBARS production. Nevertheless, the results for Mg suspensions appear to differ: while similar cell growth inhibition results were recorded in the presence of soluble and structural Mg, that was not the case for {TBARS}. These opposing ndings are thought to be caused because plausible cell regeneration after LP (Dogterom et al., 1989). In particular, we do not discard the possibility that, for experimental conditions for when LP levels (as determined {TBARS}) were found to be low, cell regeneration could have taken place. Further work is being conducted to test this hypothesis. Acknowledgements The authors thank Lic Maria del Roco Galindo Ortega (UAMCuajimalpa) for technical assistance. This project was supported in part by Universidad Autonoma Metropolitana Unidad Cuajimalpa and ECACORE 2020 (SEMARNAT CONACYT 23496). References
Altura, B.M., Gebrewold, A., Zhang, A., Altura, B.T., 2003. Low extracellular magnesium ions induce lipid peroxidation and activation of nuclearfactorkappa B in canine cerebral vascular smooth muscle possible relation to traumatic brain injury and strokes. Neurosci. Lett. 341, 189192. Babizhaev, M.A., 1988. The biphasic effect of calcium on lipid peroxidation. Arch. Biochem. Biophys. 266, 446451. Beatrice, M.C., Stiers, D.L., Pfeiffer, D.R., 1984. The role of glutathione in the retention of Ca2+ by liver mitochondria. J. Biol. Chem. 259, 12791287.

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