658 H.

TURKEZ
JOURNAL OF APPLIED TOXICOLOGY
J. Appl. Toxicol. 2008; 28: 658–664
Published online 16 November 2007 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/jat.1318

Effects of boric acid and borax on titanium dioxide

genotoxicity
Hasan Turkez*
Department of Biology, Faculty of Arts and Sciences, Atatürk University, 25240, Erzurum, Turkey
Received 16 July 2007; Revised 29 September 2007; Accepted 1 October 2007

ABSTRACT: Titanium dioxide (TiO2) is a potential carcinogenic/mutagenic agent although it is used in many areas
including medical industries and cosmetics. Boron (as boric acid and borax) has also well-described biological effects and
therapeutic benefits. In a previous study, sister-chromatid exchanges (SCEs) and micronuclei (MN) rates were assessed
in control and TiO2-treated (1, 2, 3, 5, 7.5 and 10 μM) human whole blood cultures. The results showed that the rates of
SCE (at 2, 3, 5, 7.5 and 10 μM) and MN (at 5, 7.5 and 10 μM) formation in peripheral lymphocytes were increased sig-
nificantly by TiO2 compared with the controls. The present study also investigated the genetic effects of boric acid and
borax (2.5, 5 and 10 μM) on cultures with and without TiO2 addition. No significant increase in SCE and MN frequen-
cies were observed at all concentrations of boron compounds. However, TiO2-induced SCE and MN could be reduced
significantly by the presence of boric acid and borax. In conclusion, this study indicated for the first time that boric acid
and borax led to an increased resistance of DNA to damage induced by TiO2. Copyright © 2007 John Wiley & Sons, Ltd.

KEY WORDS: boric acid; borax; human lymphocytes; micronuclei; sister-chromatid exchange; titanium dioxide

Introduction free radical generation could be central for TiO2 patho-

genicity (Donaldson et al., 1996). In fact, oxidative stress
Boric acid and borax are found in an array of consumer increased after inhalation of TiO2 in human and rat alveolar
goods including fireproofing for fabrics and wood, insec- macrophages (Rahman et al., 1997). Again, cultured mouse
ticides, and in many cosmetics and personal care products brain microglia responded to TiO2 with cellular and
as well (Moore, 1997). Titanium dioxide (TiO2) is also morphological expression of free radical formation (Long
widely used in the cosmetics, pharmaceutical, paint and et al., 2006). The impairment of endothelium-dependent
paper industries (Gelis et al., 2003). In addition, TiO2 is dilation in the systemic microcirculation coincided with
used for sterilization of waste water (Wamer et al., 1997). polymorphonuclear leukocyte (PMNLs) adhesion, myelo-
Previously, TiO2 was considered to be biologically inert peroxidase (MPO) deposition and local oxidative stress
(Lindenschmidt et al., 1990). DuPont (1985) showed dose- in rats which were intratracheally instilled with TiO2
related effects of TiO2 on rats including inflammation and (Nurkiewicz et al., 2006). Moreover, the results demon-
pulmonary damage. Again, a previous study reported that strated that TiO2 could catalyse oxidative damage to DNA
exposure to particulate matter of diesel exhaust was gen- in cultured human fibroblasts (Rosemary et al., 1997).
erally considered innocuous but titanium dioxide caused Thus, serious diseases, such as cancer and arteriosclerosis,
lung tumor formations in rats (Hesterberg et al., 2005). occurred when the survival mechanisms (antioxidants)
To assess the adverse effects of TiO2 on animal and human were unable to deal adequately with the reactive oxygen
cells, a few studies have been presented (Bernard et al., species (ROS) in the cells (Valko et al., 2006). It is
1990; Schapira et al., 1995). Hepatic injury and renal lesions known that cellular antioxidant activities arise from small
were recorded in female mice (Jiangxue et al., 2006). In molecular antioxidants, such as glutathione, and anti-
addition, cystic and keratinizing squamous lesions were oxidant scavenging enzymes, such as cellular SOD and
characterized in rat lungs (Carlton, 1994). Since the species CAT (Kedziora-Kornatowska et al., 2004).
differ, most causes of pathological effects are not well Although increasing evidence shows the nutritional
established (Lindenschmidt et al., 1990; Bermudez et al., benefits of boron compounds and their ability to streng-
2004). The genotoxicity of TiO2 also remains controver- then the tissue antioxidant defenses in animals and humans
sial (Lu et al., 1998; Turkez and Geyikoglu, 2007). However, (Hunt, 1994; Pawa and Ali, 2006), the action mechanisms
of these compounds have not been clearly identified. It
is reasonable to hypothesize that sufficient boron intake
(as boric acid) causes a simple reduction in leukocyte
* Correspondence to: Hasan Turkez, Ataturk Universitesi, Fen-Edebiyat
Fakultesi, Biyoloji Bolumu, 25240, Erzurum, Turkey. ROS generation. Thus, efforts are underway to confirm
E-mail: [email protected] the corollary of the boron-inflammation hypothesis that

Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2008; 28: 658–664
DOI: 10.1002/jat
 TiO2 TOXICITY AND BORON COMPOUNDS 659

boron limits oxidative damage by enhancing body stores Leonorenstr. 2-6.D-12247, Berlin) with 5 μg ml−1 of phyto-
of glutathione and its derivatives or by inducing other hemagglutinin (Biochrom). TiO2 (CAS No. 13463-67-7)
ROS neutralizing agents (Hunt and Idso, 1999). Besides, (Sigma Chemical Co., St Louis, MO. USA) (in concen-
there is emerging evidence that borax partly normalizes trations of 1, 2, 3, 5, 7.5 and 10 μM) and two boron
the liver by modulating oxidative stress parameters (Pawa compounds: boric acid (H3BO3, CAS No. 10043-35-3)
and Ali, 2006). Again, boron supplementation significantly and borax (Na2B4O710H2O, CAS No. 1303-96-4)) (Eti
increases erythrocyte SOD activity in men and postmeno- Mine Works General Management, Turkey) were added
pausal women with a marginal copper status (Nielsen, to the cultures (in concentrations of 2.5, 5 and 10 μM)
1989). Boron deficient Anabaena PCC 7119 heterocysts just before incubation, separately and together. Thus,
also exhibit increased concentrations of SOD, catalase and the experiments were performed on different groups
peroxidase, a situation thought to arise as a consequence as follows: TiO2 alone, boric acid alone, borax alone,
of alterations in the heterocyst envelope with subsequent TiO2 + boric acid and TiO2 + borax. These investigations
facilitated O2 diffusion and an increase in ROS (Garcia- stem from the works of Lu et al. (1998) and Turkez
Gonzalez, 1991). However, it remains to be determined and Geyikoglu (2006). Each individual lymphocyte cul-
whether SOD activity increased because boron induced ture without TiO2 and boric acid or borax was studied
free radical formation, or whether boron improved anti- as a control group. For the studies, a stock solution of
oxidative activity (Hunt and Idso, 1999). the TiO2 was prepared with sterile dimethyl sulfoxide
Several studies examining the mutagenic properties of (DMSO).
boric acid, reported that boric acid had a minimal poten-
tial for genotoxicity in bacteria and cultured mammalian
cells (Moore, 1997). Basic toxicity information often pro- Sister Chromatid Exchange (SCE) Test
vides a valuable perspective for predicting the potential
risk to humans. To be clear in this information, the genetic With the aim of providing successive visualization of
actions of boron compounds on different cells need sup- SCEs, 5-bromo-2′-deoxyuridine (Sigma, final concentra-
porting studies. Blood offers several advantages as a test tion 20 μM) was added after culture initation. The cultures
system (Lerda et al., 2005). Easy availability of large were incubated in complete darkness for 72 h at 37 °C.
numbers of human cells: a few millilitres of peripheral Exactly 70 h and 30 min after beginning the incubations,
blood can easily and repeatedly be obtained from an in- colcemid (Sigma, St Louis) was added to the cultures to
dividual. A proportion of the lymphocytes can be stimu- achieve a final concentration of 0.5 μg l−1. After hypotonic
lated by mitogens to undergo mitosis in culture; they are treatment (0.075 M KCl) followed by three repetitive cycles
easy to culture and provide a ready source of dividing of fixation in methanol/acetic acid solution (3:1, v/v),
cells for in vitro induced chromosome structural changes. centrifugation and resuspension, the cell suspension was
To our knowledge, there has been no report about the roles dropped onto chilled, grease-free microscopic slides,
of boric acid and borax on titanium dioxide-treated air-dried, aged, and then differentially stained for inspec-
cultures. Thus, in this study the genetic effects of TiO2 tion of the SCE rate according to the fluorescence plus
and boron compounds on human blood were evaluated. Giemsa (FPG) procedure (Perry and Wolff, 1974). For
The objective was also to determine whether boric acid each treatment condition, 30 well-spread second division
and borax conferred protection against a wide range of metaphases containing 42–46 chromosomes in each cell
TiO2 exposure in vitro. With this aim, in the present were scored, and the values obtained were calculated as
study SCE and MN tests were performed. SCEs per cell.

Materials and Methods Micronucleus Assay

Human peripheral blood lymphocyte cultures were set up The MN test was performed by adding cytochalasin B
according to a slight modification of the protocol described (Sigma; final concentration of 6 μg ml−1) after 44 h of
by Evans and O’Riordan (1975). Whole heparinized blood culture (Fenech and Morley, 1985). At the end of the
from four healthy non-smoking donors between age 25 72-h incubation period, the lymphocytes were fixed with
and 28 with no history of exposure to any genotoxic ice-cold methanol:acetic acid (1:1). The fixed cells were
agent were used. Questionnaires were obtained for each put directly on slides using a cytospin, and stained with
blood donor to evaluate exposure history and informed May Grünwald-Giemsa. All slides were coded before
consent forms were signed by each donor. For all the scoring. The criteria for scoring micronuclei were as
volunteers hematological and biochemical parameters described by Fenech (1993). At least 2000 binucleated
were analysed and no disease was detected. lymphocytes were examined per concentration (two cul-
The heparinized blood (0.5 ml) was cultured in 5 ml of tures per concentration) for the presence of one, two
culture medium (Chromosome Medium B, Biochrom, or more micronuclei.

Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2008; 28: 658–664
DOI: 10.1002/jat
660 H. TURKEZ

Statistical Analysis

The statistical analysis of experimental values in the SCE

test was performed by one-way analysis of variance
(ANOVA) and, subsequently, by Student’s t-test and
post-hoc Dunnett’s t-test using the S.P.S.S. 11.5 software.
The statistical analysis of MN frequencies was performed
by use of the χ2 test. A P value of ≤0.05 was regarded
as indicative of statistical significance for all tests
used.

Results

According to the results, no concentration of boric acid

and borax alone significantly affected SCE rates and MN
frequencies in human blood cultures. Also, no significant
increases in SCE and MN frequencies were observed
at the lowest TiO2 dose (1 μM) (data not shown). On the
contrary, increasing concentrations of TiO2 significantly
elevated SCEs (2, 3, 5, 7.5 and 10 μM) and MN (5, Figure 2. The MN rates in blood lymphocytes after
7.5 and 10 μM) in lymphocytes compared with controls exposure to different doses of TiO2 and boron com-
(Figs 1 and 2). pounds. Abbrevations are as in Figure 1
Whereas, boric acid and borax (at all concentrations)
reduced the number of TiO2-induced SCE and MN for-
mations. However, boron compounds did not completely
inhibit TiO2-caused SCE and MN induction (5, 7.5 and Discussion
10 μM) (Figs 3 and 4).
SCEs are often included as a genotoxic endpoint to reflect
DNA damage, or as a biological dosimeter or biomarker
of exposure (Spronck and Kirkland, 2002). Several different
mechanisms may be involved in the formation of MN,
including mitotic spindle disruption (aneugenic) or chro-
mosome breakage (Heddle et al., 1983; Yuzbasioglu et al.,
2006). In the previous study, SCEs and MN were in-
creased in lymphocytes treated with TiO2 alone and these
effects were highly magnified with TiO2 dosage (Turkez
and Geyikoglu, 2007). Note in this context that TiO2
caused SCEs in Chinese hamster ovary-K1 cell cultures
(CHOK1) (Lu et al., 1998). Again, similar findings were
reported by other authors: Shelby et al. (1993) reported
the induction of MN in the bone marrow of mice and
Rahman et al. (2002) showed that TiO2 induces micro-
nuclei in Syrian hamster embryo (SHE) fibroblasts. However,
Miller et al. (1995) showed that TiO2 did not induce MN
formation in CHO-K5 cells. The mechanisms underlying
TiO2 induction of MN and SCEs are still unclear. Studies
have shown that the pathological alterations induced by
TiO2 exposure may be associated with the generation of
hydroxyl radicals (Bernard et al., 1990; Schapira et al.,
1995) or may not be (Olmedo et al., 2005). On the other
Figure 1. The SCE rates in blood lymphocytes after hand, Wamer et al. (1997) reported that nucleic acids
exposure to different doses of TiO2 and boron com- are especially potential targets for oxidative damage sen-
pounds. BA, boric acid; BX, borax; means in the figure sitized by TiO2. Again, evidence in vitro and in vivo
followed by the different letters present significant indicate that increasing oxidative stress causes DNA
differences at the P < 0.05 level damage (Nagy et al., 2005).

Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2008; 28: 658–664
DOI: 10.1002/jat
 TiO2 TOXICITY AND BORON COMPOUNDS 661

Figure 3. The positive effects of boric acid and borax on the number of TiO2-induced SCE formations. Abbreva-
tions are as in Figure 1

The study also revealed that the dramatic reduction Treatment with borax could cause reductions in the activ-
of TiO2-induced SCEs and MN formations were due to ity levels of some pro-oxidant enzymes in the liver (e.g.
the protective roles of boric acid and borax. It is known xanthine oxidase), consequently ameliorating the tissue
that TiO2 has pro-oxidant effects (Afaq et al., 1998). damage (Pawa and Ali, 2006). There is no evidence on

Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2008; 28: 658–664
DOI: 10.1002/jat
662 H. TURKEZ

Figure 4. The positive effects of boric acid and borax on the number of TiO2-induced MN formations. Abbreva-
tions are as in Figure 1

the activities of present enzymes in the blood by boron factors lead to antioxidant depletion (Hedenborg, 1988),
compounds in this investigation. However, these com- thus, chemical-induced genotoxicity occurs, including
pounds could strengthen the antioxidant defense mecha- oxidation of purine nucleotides of nucleic acids and that
nism as mentioned above against TiO2. All pro-oxidant of protein SH groups (Hooiveld et al., 1998; Xi et al.,

Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2008; 28: 658–664
DOI: 10.1002/jat
 TiO2 TOXICITY AND BORON COMPOUNDS 663

2004). In conclusion, defective DNA repair and DNA properties of these agents is difficult to understand. The
damage form SCEs (Bozkurt et al., 2003). At this point, results also showed that boric acid and borax did not
it is important to establish DNA damage. As a matter of completely inhibit induction of SCE and MN formation
fact, DNA damage is a useful biomarker of the oxidative by TiO2.
status and the antioxidant defense system (Duthie et al.,
Acknowledgements—We are grateful to all the volunteers for the blood
1996). samples. The author is grateful to Savas Yesilyurt for the linguistic
Reactive oxygen species formation and lipid peroxida- support in preparing the article.
tion (LPO) induced by TiO2 might play a main role in its
cytotoxicity (Gurr et al., 2005). Pawa and Ali (2006) re-
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