ARTICLE
http://www.ufrgs.br/seerbio/ojs ISSN 1980-4849 (on-line) / 1679-2343 (print)
Genotoxic potenti��� o�� t�e �i�� in en�i��on�ent�� �it� p�otocopie����
otenti��� o�� t�e �i�� in en�i��on�ent�� �it� p�otocopie����
�it� p�otocopie����
ABSTRACT: (�enoto�ic potential of the air in environ�ents with photocopiers). The conta�inants �ost co��onl�� found in�enoto�ic potential of the air in environ�ents with photocopiers). The conta�inants �ost co��onl�� found inwith photocopiers). The conta�inants �ost co��onl�� found in environ�ents with photocopiers are the volatile organic co�pounds (VOCs), aro�atic pol��c��clic h��drocarbons, aldeh��des, ketones, ozone, and particles. The present stud�� ai�ed at the assess�ent of the genoto�icit�� of the air in environ�ents with photocopiers. Si� clones BNL 4430 of Tradescantia h��brid (T. subcaulis Bush � T. hirsutifolia Bush) had been kept for 15 da��s in two environ�ents: one of the� with four photocopiers working 12 hours per da�� (treat�ent 1), and the other with three photocopiers working eight hours per da�� (treat�ent 2). Other si� plants co�prised the check group and were kept in a greenhouse. The frequenc�� of genic and chro�oso�al �utations was deter�ined b�� assess�ent of the sta�en hair cells and tetrads, after 15 da��s of e�position. There were quantified 415 sta�en hairs and 1,500 tetrads per treat�ent. The plants fro� treat�ent 1 have showed significant differences in relation to the check group (p≤0.05) to the frequenc�� of chro�oso�al da�age. No significant difference was observed in the sta�en hairs for genic �utation in relation to the check group.
Key �o��d��: chro�oso�al �utation, genic �utation, indoor genoto�icit��, photocopier, toner, Tradescantia.
RESUMO: (Potencial genotó�ico do ar e� a�bientes co� fotocopiadoras). Os conta�inantes �ais co�u�ente encontrados e�
a�bientes co� fotocopiadoras são os co�postos orgânicos voláteis (VOCs), hidrocarbonetos policíclicos aro�áticos, aldeídos, cetonas, ozônio e partículas. O presente estudo teve co�o objetivo avaliar a genoto�icidade do ar e� a�bientes co� fotoco-piadoras. Seis clones híbridos BNL 4430 of Tradescantia (T. subcaulis Bush � T. hirsutifolia Bush) fora� �antidos durante 15 dias e� dois a�bientes: u� deles contendo quatro �áquinas fotocopiadoras que funciona� durante 12 horas/dia (trata�ento 1) e outro co� três �áquinas, que funciona� durante 8 horas/dia (trata�ento 2). Outras seis plantas constituíra� o grupo controle e fora� �antidas e� u�a casa de vegetação. A frequência de �utações gênicas e cro�ossô�icas foi deter�inada a partir de cinco avaliações das células do pêlo esta�inal e tétrades, após 15 dias de e�posição. Fora� quantificados 415 pêlos esta�inais e 1.500 tétrades por trata�ento. As plantas do trata�ento 1 apresentara� diferenças significativas e� relação ao grupo controle (p≤0,05) para a frequência de danos cro�ossô�icos. Nos pêlos esta�inais não fora� observadas diferenças significativas para as �utações gênicas e� relação ao grupo controle.
P����������-c���e: fotocopiadoras, genoto�icidade indoor, �utação cro�ossô�ica, �utação gênica, toner, Tradescantia.
�edalva Terezinha Ribeiro Filipini1, André Búrigo Leite1 and Vânia Helena Techio1*
1. Universidade do Contestado, UnC. Rua Vitor Sopelsa, 3000, CEP 89700-000, Concórdia, SC, Brazil. * Author for correspondence. E-�ail: vaniatechio@��ahoo.co�.br.
Received: March 23 2009 Received after revision: Ma�� 25 2009 Accepted: August 12 2009 Available online: http://www.ufrgs.br/seerbio/ojs/inde�.php/rbb/article/view/1194
INTRODUCTION
The qualit�� of the air in the indoor environ�ent is a new area of research and has attracted attention for de�onstrating the positive association between the indoor pollutant sources and the worsening of health (�ioda & Aquino Neto 2003, Saldiva & Miraglia 2004). The indoor concentration of �an�� pollutants is superior to the outdoor concentration, because the polluting sources are confined in a space that hinders the dispersion and increases the risk to health of the individual e�posed.
A�ong the usuall�� �onitored conta�inants in the indoor environ�ents, there are the carbon dio�ide and �ono�ide, total volatile, organic, and se�i-volatile organic co�pounds, particulate �atter, nicotine, and �icroorganis�s (�ioda & Aquino Neto 2003). Specificall�� the environ�ent with photocopiers is identified as high priorit�� in the research for controlling the indoor conta�ination. The �achines, besides producing electro�agnetic fields, are know as sources of volatile organic co�pounds (VOCs), aro�atic pol��c��clic h��drocarbons, aldeh��des, ketones, ozone, and particulate �atter (Hetes et al. 1995, Northein
et al. 1998, Brickus & Aquino Neto 1999, California
Environ�ental Protection Agenc�� 2005). The e�ission of che�ical co�pounds �a�� happen as a direct result of �achine operation or fro� its co�ponents such ink and toner. The toner is co�posed b�� resins (pol��st��rene, pol��ester, pol��eth��lene, epo���, pol��prop��lene), black carbon, �agnetite, silica, nitrop��rene, benzene, and aro�atic pol��c��clic co�pounds (Lofroth et al. 1980, Rosenkranz et al. 1980).
There are several evidences showing that �agnetic fields and co�ponents of the toner, individuall�� or in co�ple� �i�tures, are genoto�ic (Rosenkranz et al. 1980, Iravath�� �oud et al. 2001, Iravath�� �oud et al. 2004, Valberg et al. 2006, Brod�� et al. 2007). The co�position, co�ple�it��, and availabilit�� of these co�pounds depend on the size, nu�ber, and �aintenance of the �achines, the a�ount of copies obtained, di�ensions of the environ�ent where the�� are places, ventilation, air renewing, and the presence of filters for purif��ing the air (Hetes et al. 1995).
The �onitoring of the e�posure to these substances �a�� be acco�plished through the assess�ent of the biological and che�ical para�eters. In general, the che�ical anal��ses de�and sophisticated and e�pensive equip�ents, besides not offering directl�� the infor�ation
regarding the threat to the biological s��ste�s. The assa��s with the ani�al and vegetal species, or bioindicators, are interesting alternatives due to their low cost, rapid, and reliable results. Although, the tests using ani�als are preferred for stud��ing the air conta�inants (Saldiva & Böh� 1998), the plants of the genus Tradescantia have shown to be a suitable �odel for the genoto�icit�� studies (�ui�arães et al. 2004, Misik et al. 2006). According to the U.S. Environ�ental Protect Agenc�� (1980), the assa��s with plants are �ore sensitive than other s��ste�s regarding the detection of genoto�ic co�pounds present in the environ�ent. The present stud�� ai�ed at the assess�ent of the genoto�icit�� of the air in two environ�ents with photocopiers using the sta�en hairs and the �icronucleus bioassa�� in clones BNL4430 of Tradescantia h��brid (T. subcaulis Bush �
T. hirsutifolia Bush).
MATERIAL AND METHODS
In situ exposition
The assess�ent of genoto�icit�� was carried in two environ�ents. The treat�ent 1 was perfor�ed in area of 6.76 � × 7.89 �, projecting windows disposed in two entire walls and four photocopiers working 12 hours per da��. The treat�ent 2 was in area of 6.74 � × 7.89 �, it has ventilation flaps occup��ing two walls entirel�� and presents three photocopiers working 8 hours per da��. In both environ�ents, there is no s��ste� of e�haustion or filters.
�roups of si� pots containing clones BNL 4430 of
Tradescantia h��brid (h��brid (T. subcaulis Bush � T. hirsutifolia Bush), donated b�� the Co�issão Nacional de Energia, donated b�� the Co�issão Nacional de Energia Nuclear de Poços de Caldas, Minas �erais State, Brazil, were e�posed for 15 da��s in both places. Si�ultaneousl��, the plants of the control group were placed in greenhouse under controlled conditions. The substrate used in the pots is an organic co�pound (Ca�psulfértil®) obtained after several fer�entations, followed b�� sterilization. The greenhouse and the two environ�ents are located at Universidade do Contestado–UnC, in Concórdia, Santa Catarina State, Brazil.
Micronucleus MN) and stamen hair (TRAD-SHM) bioassay
After the e�position, ��oung buds were collected and
fi�ed in a solution of eth��lic alcohol: acetic acid (3:1, v:v) and spared at -4 ºC until the �icronuclei anal��ses of the tetrads (TRAD-MN). Other floral branches were placed under h��droponic s��ste� for anal��sis of the sta�en hair cells (TRAD-SHM).
The procedures for deter�ining the �utagenicit�� were done according to Rodrigues (1999a,b), Ichikawa (1992) and Ma (1990). The frequenc�� of �utations was deter�ined based on the five assess�ents perfor�ed in 415 sta�en hairs and 1,500 tetrads per treat�ent. The assess�ents of the sta�en hairs allowed the detection of genic �utations, and the assess�ents of the �icronuclei in tetrads were indicative of chro�oso�al �utations.
The �agnitude of the �icronuclei and genic �utations variations were esti�ated b�� variance anal��sis followed b�� the Tuke��’s test. The anal��ses were perfor�ed using the Software SisVar (Ferreira 2003).
RESULTS AND DISCUSSION
The BNL 4430 clones used in the present stud�� have de�onstrated to be a suitable �odel for anal��sing the �utagenicit�� in indoor environ�ents, corroborating with previous studies of Ma & Harris (1987a,b) and Alves et
al. (2003).
The plants fro� treat�ent 1 e�hibited rates of chro�oso�al da�age (presence of �icronuclei) with significant differences in relation to control group (p≤0.05) (Tab. 1, Fig. 1A). Regarding the sta�en hairs, independentl�� of the environ�ent anal��sed, there was no significant difference for the genic �utation in relation to the control group (Tab. 2). The Table 2 presents the sa�e sta�en hairs features described b�� Ichikawa (1992), which de�onstrated the absence of consequences upon the plant’s flora structure.
The �icronuclei are for�ed during the telophase, when the nuclear envelope is reconstituted around the chro�oso�es of the daugther-cells. These �icronuclei �a�� have been a result of acentric chro�oso�al frag�ents or entire chro�oso�es that were not included in the �ain nucleus due to structural da�ages (Fenech 2000), as consequence of action of ph��sical, che�ical or biological agents (Fenech 2000, Stopper & Müller 1997, Kirsch-Volders et al. 1997).
The co�pounds under dispersion in these environ�ents were able to induce ger�inative �utations, but have not e�erted significative effect on the so�atic tissue, as the T�b��e 1. Mutation rate ± SD (�utant events nu�bers/300 tetrads) detected b�� the bioassa�� of �icronucleus in pollen �other cells (TRAD-MN)
of clones BNL 4430 of Tradescantia h��brids (T. subcaulis Bush � T. hirsutifolia Bush) e�posed to the photocopiers in the two environ�ents and in the greenhouse
A����e�����ent�� En�i��on�ent 1 En�i��on�ent 2 G��een�ou��e (cont��o��)
1 82±2.83 34±1.30 0
2 69±3.12 68±2.91 4±0.30
3 124±4.70 31±1.12 0
4 17±1.12 21±1.01 0
5 45±1.93 65±3.02 0
Mean 67.4a 43.8ab 0.8b
da�ages in leukoc��tes of the photocopier’s operators. In the stud��, there were observed the significative increase in the rate of �utation when the individuals were assessed after 15 and 30 �inutes fro� the beginning of the e�position, which decreased graduall�� after 45 �inutes. According to the authors, this increase happened due to the breakages in one of the DNA strands that were converted while the cell passed through repair process, pointing therefore the interaction between the co�ple� che�ical �i�tures and the DNA.
According to Carvalho (2005), the nu�erous si�ilarities between the genetic constitution of superior plants and �a��als �a�� suggest si�ilar effects of a �utagen upon the plant and �a��al’s DNAs. However, there are organizational and ph��siological differences between the�, �ainl�� in the �orphogenesis and �etabolis�. These differences �a�� be a result of different reactions if considered a chro�oso�al lesion. In the hu�an being, e.g., onl�� a �ini�al fraction upon the DNA �a�� or �a�� not lead to �utations, while in
Tradescantia �ost of the lesions results in �utation.
A deter�ined increase of the frequenc�� on the initial da�age will increase in the sa�e proportion the �utation incidence rate in both hu�an being and Tradescantia (Ichikawa 1992, Ichikawa 1997). Thus, a relative increase in the so�atic �utation frequenc�� in Tradescantia �a�� indicate a proportional increase of the risk of �utation for hu�an beings (Carvalho 2005).
Several authors describe the �utagenic and carcinogenic effects of so�e che�ical substances found in the toner sta�en hair. Previous studies regarding the geno�e of
the Tradescantia species acco�plished b�� de Sa� and Ed�onds (1933), Steinitz (1944) and Underbrink et
al. (1973) have alread�� de�onstrated that the �eiotic
chro�oso�es are �ore susceptible to the breakage than the others under rest. According to UnderbrinkAccording to Underbrink et al. (1973), both tests are e�tensivel�� used to detect �utations in Tradescantia. However, the efficienc�� of �icronucleus test displa��s appro�i�atel�� 36 ti�es greater. Stud��ing the effects of deficienc�� in �agnesiu� (Mg), sulfur (S) and calciu� (Ca) in Tradescantia paludosa, Steffensen (1953, 1954 and 1955) also described a greater sensitivit�� of �icrospores co�pared with root tips, according to preli�inar�� evidence of increased susceptibilit�� of �eiotic to �itotic cells.
The highest sensitivit�� of the �eiotic chro�oso�es is guaranteed b�� the lowest specificit�� of the da�age necessar�� to produce �icronuclei when co�pared to the �utation in the sta�en hairs. In this case, it is possible to assu�e that nu�erous loci in each one of the 12 chro�oso�es of BNL 4430 clones is subjected to breakages, which lead to the for�ation of �icronuclei. Oppositel��, in the sta�en hairs, the �utation �a�� occur in a specific locus in a chro�oso�e for the production of pink cells (Ma et al. 1978).
Other e�planation to a lower rate of so�atic �utation is that the cells present a repair s��ste� for �utation that �akes possible its reversion after 15 da��s of e�position. The DNA repair s��ste� was alread�� observed b�� Iravath�� �oud et al. (2001), when assessing the chro�oso�al
T�b��e 2. S��ste� of sta�en hairs and �utant events ± SD (in 415 sta�en hair) of clones BNL 4430 of Tradescantia h��brids (T. subcaulis Bush
� T. hirsutifolia Bush) e�posed to the photocopiers in the two environ�ents and in the greenhouse.
C�����cte��i��tic�� En�i��on�ent 1 En�i��on�ent 2 G��een�ou��e (cont��o��)
Mean of hairs per sta�en 41.2±1.12 41.9±1.63 41.2±0.93
Mean of cells per hairs 25.55±0.90 25.86±0.98 25.55±0.70
Esti�ation of hairs per flower 247.2±4.20 251.4±3.94 247.2±3.63
Nu�ber of �utant events 0a 2a±0.27 0a
*Nu�bers in the row followed b�� the sa�e letter do not differ statisticall�� b�� the Tuke��’s test (p≤0.05)
Figu��e 1. Pink �utant cell (arrow) in sta�en hair (A) and tetrad with �icronucleus (B, arrow) in clones BNL 4430 of Tradescantia h��brids
such as st��rene that is able to induce chro�oso�al �utation and change a�ong the sister chro�atids (Vaghef & Hell�ann 1998), benzene that causes breakages in one of the DNA strands (Andreoli et al. 1997), and the aro�atic pol��c��clic ones that cause o�idative da�ages and induce breakage in the DNA (Popp et al. 1997).
The results found here have shown that the bioassa�� of BNL 4430 clones �a�� be used in the qualit�� control of the indoor air in environ�ents with photocopiers. The plants e�posed to the environ�ent with a higher photocopiers nu�ber and during a longer functioning ti�e have presented higher genetic da�age rates. A�ong the�, the predo�inance of chro�oso�al �utations indicates that there were a high sensibilit�� to the PMCs during the detection of �utagens present in the air. Moreover, the results reinforce the need of investigation and identification regarding the search of wa��s to reduce and control of the substances e�ission, which �a�� induce �utations.
REFERENCES
ALVES, E.S.; PEDROSO, A.N.V.; DOMIN�OS, M.; �UIMARÃES, E.T.; SALDIVA, P.H.N. 2003. Bio�onitora�ento indoor do potencial �utagênico e� laboratórios e herbário do Instituto de Botânica por �eio do bioensaio Trad-MCN. Hoehnea, 30: 1-2.
ANDREOLI, C.; LEOPARDI, P.; CREBELLI, R. 1997. Detection of DNADetection of DNA da�age in hu�a� l���phoc��te b�� alkaline single cell gel electrophoresis after e�posure to benzene or benzene �etabolites. Mutation Research, 377: 95-104.
BRICKUS, L.S.R. & AQUINO NETO, F.R. 1999. A qualidade do ar de interiores e a quí�ica. Química Nova, 22: 65-74.
BRODY, J.; KAVANAU�H-LYNCH, M.H.E.; OLOPADE, O.I.; PALMER, W.L.; SHINA�AWA, S.M.; STEIN�RABER, S.; WILLIANS, D.R. 2007. Identifying gaps in breast cancer research: adressing disparities and the roles of the physical and social development. California: California BreastCalifornia: California Breast Cancer Research Progra�. 5 p.
CALIFORNIA ENVIRONMENTAL PROTECTION A�ENCY. 2005. Indoor air pollution in California. California. 363 p.
CARVALHO H. A. 2005. A Tradescantia co�o bioindicador vegetal na �onitoração dos efeitos clastogênicos das radiações ionizantes. Radiologia Brasileira, 38: 459-462.
FENECH, M. 2000. The in vitro �icronucleus technique. Mutation Research, 455: 81-95.
FERREIRA, D. F. 2003. Sisvar®. Versão 4.6 (Build 61);Universidade Federal de Lavras, Brasil.
�IODA, A. & AQUINO NETO, F.R. 2003. Poluição quí�ica relacionada ao ar de interiores. Química Nova, 26: 359-365.
�UIMARÃES, E.T.; MACCHIONE, M.; LOBO, D.J.A.; DOMIN�OS, M.; SALDIVA, P.H.N. 2004. Evaluation of the �utagenic potential of urban air pollution in São Paulo, Southeastern Brazil, using the Tradescantia sta�en-hair assa��. Environment Toxicology, 19: 578-584.
HETES, R.; MOORE, M.; NORTHEIM, C. 1995. Office equipment: design, indoor air emissions, and pollution prevention opportunities. USA: Environ�ental Protection Agenc��- research and develop�ent. 4 p. ICHIKAWA S. 1992. Tradescantia sta�en-hair s��ste� as an e�cellent botanical tester of �utagenicit��: it’s responses to ionizing radiationsl tester of �utagenicit��: it’s responses to ionizing radiations and che�ical �utagens and so�e s��nergistic effects found. Mutation Research, 270: 3-22.
ICHIKAWA, S. 1997. So�atic �utation frequencies in Tradescantia sta�en hairs treated with relativel�� low ther�al neutron flu�es.ivel�� low ther�al neutron flu�es. Radiation Research, 147: 109-114.
IRAVATHY �OUD, K.; SHANKARAPPA, K.; VIJAYASHREE, B.; PRABHAKAR RAO, K.; AHUJA, Y.R. 2001. DNA da�age and repair studies in individuals working with photocop��ing �achines. Indian Journal of Human Genetics, 1: 139-143.
IRAVATHY �OUD, K.; HASAN, Q.; BALAKRISHNA, N.; PRABAKHAR RAO, K.; AHUJA, Y. R. 2004. �enoto�icit�� evaluation of individuals working with photocop��ing �achines. Mutation research, 563: 151-158. KIRSCH-VOLDERS, M.; ELHAJOUJI, A.; CUNDARI, E.; VAN HUMMELEN, P. 1997. The in vitro �icronucleus test: a �ulti-endpoint assa�� to detect si�ultaneousl�� �itotic dela��, apoptosis, chro�oso�e breakage, chro�oso�e loss and non disjunction. Mutation Research, 392: 19-30.
LOFROTH, �.; HEFNER, E.; ALFHELM, I.; MOLLER, M. 1980. Mutagenic activit�� in photocopies. Science, 209: 1037-1039.
MA, T.H.; SPARROW, A.H.; SCHAIRER, L.A.; NAUMAN, A.F. 1978. Effect of 1,2-dibro�oethane (DBE) on �eiotic chro�oso�es of Tradescantia. Mutation Research, 58: 251-258.
MA, T.H. 1990. In situ �onitoring of environ�ental clastogens using Tradescantia-�icronucleus bioassa��. In: SANDHO, S.S. et al. (Eds) Environmental Research. New York: Plenu� Press. p. 183-190. MA, T.H. & HARRIS, M.M. 1987a. The Tradescantia �icronucleus (TRAD-MCN) assa��- a potential indoor pollution �onitoring s��ste�. Environment Mutation, 9: 65.
MA, T.H. & HARRIS, M.M. 1987b. Tradescantia �icronucleus (TRAD-MCN) bioassa�� – a pro�issing indoor air polluttion �onitoring s��ste�. In: SEIFERT, B. et al. (Eds.) 4th International Conference on indoor air quality and climate. Berlin: Institute for water, soil and air h��giene, p. 243-247.Berlin: Institute for water, soil and air h��giene, p. 243-247. MISIK, M.; SOLENSKÁ, M.; MICIETA, K.; MISIKOVÁ, K.; KNASMÜLLER, S. 2006. In situ �onitoring of clastogenicit�� of a�bient air in Bratislava, Slovakia using the Tradescantia �icronucleus assa�� and pollen abortion assa��s. Mutation Research, 605: 1-6.
NORTHEIN, C.; SHELDON, L.; WHITAKER, D.; HETES, B.; CAL�IANI, J. 1998. Indoor air emissions from office equipment: test method development and pollution prevention opportunities. USA: Environ�ental Protection Agenc��- research and develop�ent. 4 p. POPP, W.; VAHRENHOLZ, C.; SHELL, C.; �RIMMER, �.; DETTBARHN, �.; KRAUS, R.; BRAUKSIEPE, A.; SCHMELIN�, B.; �UZEIT, T.; VON BULLOW, J.; NORPHOT, K. 1997. DNA single strand breakage, DNA adducts and sister chro�atides e�changes in l���phoc��tes and phenanthrene and p��rene �etabolites in urine of coke oven workers. Occupational and Environmental Medicine, 54: 176-183.
RODRI�UES, �.S. 1999a.I�UES, �.S. 1999a. Bioensaios de toxicidade genética com plantas superiores Tradescantia (MCN e SHM), milho e soja. Jaguariúna: E�brapa Meio A�biente. 30 p.
RODRI�UES, �.S. 1999b. Bioensaios de toxicidade genética com Tradescantia. Jaguariúna: E�brapa Meio A�biente. 56 p.Jaguariúna: E�brapa Meio A�biente. 56 p.
ROSENKRANZ, H.S.; MCCOY, E.C.; SANDERS. D.R.; BUTLES, M.; KIRIAZIDES, D.K.; MERMELSTEIN, R. 1980. Nitrop��renes: isolation, identification, and reduction of �utagenic i�purities in carbon black and toners. Science, 209: 1039-1042.
SALDIVA, P.H.N. & BÖHM, �.M. 1998. Ani�al indicator of adverse effects associated with air pollution. Ecosystem Health, 4:230-235. SALDIVA, P.H.N. & MIRA�LIA, S.�.E.K. 2004. Health effects ofof cookstove e�issions. Energy for Sustainable Development, 8: 13-19. SAX, K. & EDMONDS, H.W. 1933. Develop�ent of the �ale ga�etoph��te in Tradescantia. Botanical Gazette, 95: 156-163.
STEFFENSEN, D. 1953. Induction of chro�oso�e breakage at �eiosis b�� a �agnesiu� deficienc�� in Tradescantia. Proceedings of the National Academy of Sciences, 39: 613-620.
STEFFENSEN, D. 1954. Irregularities of chro�oso�e divisions in Tradescantia grown on low sulfate. Experimental Cell Research, 6: 554-556.
STEFFENSEN, D. 1955. Breakage of chro�oso�es in Tradescantia with calciu� deficienc��. Proceedings of the National Academy of Sciences,
41: 155-160.
STEINITZ, L.M. 1944. The effect of lack of o���gen on �eiosis in Tradescantia. American Journal of Botany, 31: 428-443.
STOPPER, H. & MÜLLER, S.O. 1997. Micronuclei as a biological endpoint for genoto�icit��: a �inireview. Toxicology in vitro, 11: 661-667.
U.S. Environ�ental Protection Agenc��. 1980. Current status of bioassa��s in genetic to�icolog�� (�ene-to�). EPA, 3-5: 1-69.
VA�HEF, H. & HELLMANN, B. 1998. Detection of st��rene and st��rene
o�ide-induced DNA da�ages in various organs of �ice using the co�et assa��. Pharmacology & Toxicology, 83: 69-74.
VALBER�, P.A.; LON�, C.; SAX, S.N. 2006. Integrating studies on carcinogenic risk of carbon black: epide�iolog��, ani�al e�posures, and �echanis� of action. Journal of Occupational and Environmental Medicine, 48: 1291-1307.
UNDERBRINK, A.�.; SPARROW, A. H.; SAUTKULIS, D.; MILLS, R. E. 1975. O���gen enhance�ent ratios (OERs) for so�atic �utations in Tradescantia sta�en hairs. Radiation Botany, 15:161-168.
