<?xml version="1.0" encoding="ISO-8859-1"?><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance">
<front>
<journal-meta>
<journal-id>0034-7744</journal-id>
<journal-title><![CDATA[Revista de Biología Tropical]]></journal-title>
<abbrev-journal-title><![CDATA[Rev. biol. trop]]></abbrev-journal-title>
<issn>0034-7744</issn>
<publisher>
<publisher-name><![CDATA[Universidad de Costa Rica]]></publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id>S0034-77442013000400036</article-id>
<title-group>
<article-title xml:lang="en"><![CDATA[Toxicological effects of prolonged and intense use of mosquito coil emission in rats and its implications on malaria control]]></article-title>
<article-title xml:lang="es"><![CDATA[Efectos toxicológicos del uso prolongado e intenso de emisiones de espirales contra mosquitos en ratas y sus implicaciones sobre el control de la malaria]]></article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Taiwo Idowu]]></surname>
<given-names><![CDATA[Emmanuel]]></given-names>
</name>
<xref ref-type="aff" rid="A01"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Aimufua]]></surname>
<given-names><![CDATA[Oyenmwen Judith]]></given-names>
</name>
<xref ref-type="aff" rid="A01"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Yomi-Onilude]]></surname>
<given-names><![CDATA[Ejovwoke]]></given-names>
</name>
<xref ref-type="aff" rid="A01"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Akinsanya]]></surname>
<given-names><![CDATA[Bamidele]]></given-names>
</name>
<xref ref-type="aff" rid="A01"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Adetoro Otubanjo]]></surname>
<given-names><![CDATA[Olubumi]]></given-names>
</name>
<xref ref-type="aff" rid="A01"/>
</contrib>
</contrib-group>
<aff id="A01">
<institution><![CDATA[,University of Lagos  ]]></institution>
<addr-line><![CDATA[ Lagos]]></addr-line>
<country>Nigeria</country>
</aff>
<pub-date pub-type="pub">
<day>00</day>
<month>09</month>
<year>2013</year>
</pub-date>
<pub-date pub-type="epub">
<day>00</day>
<month>09</month>
<year>2013</year>
</pub-date>
<volume>61</volume>
<numero>3</numero>
<fpage>1463</fpage>
<lpage>1473</lpage>
<copyright-statement/>
<copyright-year/>
<self-uri xlink:href="http://www.scielo.sa.cr/scielo.php?script=sci_arttext&amp;pid=S0034-77442013000400036&amp;lng=en&amp;nrm=iso"></self-uri><self-uri xlink:href="http://www.scielo.sa.cr/scielo.php?script=sci_abstract&amp;pid=S0034-77442013000400036&amp;lng=en&amp;nrm=iso"></self-uri><self-uri xlink:href="http://www.scielo.sa.cr/scielo.php?script=sci_pdf&amp;pid=S0034-77442013000400036&amp;lng=en&amp;nrm=iso"></self-uri><abstract abstract-type="short" xml:lang="en"><p><![CDATA[Efectos toxicológicos del uso prolongado e intenso de emisiones de espirales contra mosquitos en ratas y sus implicaciones sobre el control de la malaria. Mosquito coil is a vector control option used to prevent malaria in low income counties, while some studies have addressed this issue, additional reseach is required to increase knowledge on the adverse health effects caused by the prolonged use of coils. In this study we investigated the toxicological effects of fumes from two locally manufactured mosquito coil insecticides (with pyrethroids: transfluthrin and d-allethrin as active ingredients) on male albino rats. For this, we recorded the haematological and biochemical indices, and made histopathology and mutagenicity evaluations in rats exposed to mosquito fumes during 2, 4, 8, 12 and 16 week periods. Haematological determination was performed using automated hematology analyzer to determine White Blood Cell (WBC), Packed Cell Volume (PCV), Red Blood Cell (RBC) and Platelet (PLT) counts, while biochemical evaluations were determined using available commercial kits. Gross histopathological changes were studied for the kidney, liver and lungs in sacrificed rats. The rat sperm head abnormalities assessment was used to evaluate mutagenicity. Mosquito coil fumes produced significant increase (P<0.05) in the levels of total protein, total albumin and bilirubin, when animals were exposed from two weeks to 16 weeks with transfluthrin. Similarly, elevation in the activities of aspartate amino transferase, alanine amino transferase and alanine phosphatase, increased significantly in both insecticides. Increase in WBC, RBC and PCV were recorded for all the exposure periods, however PLT count showed no significant increase (P&gt;0.05). Mutagenicity assessment revealed sperm abnormality was statistically significant (P<0.05) compared with the control at 8, 12 and 16 weeks post exposure to transfluthrin. Histological studies revealed severe lung damage evidenced by interstitial accumulations, pulmonary oedema and emphysema in exposed rats. Intracellular accumulations and severe sinusoidal congestion of liver cells were observed from 12 weeks exposure, indicating liver damage. Our studies indicate that mosquito coil fumes do initiate gradual damage to the host. These pathological effects must be taken into consideration by the malaria control program, particularly when regulating their long term and indoor usage.]]></p></abstract>
<abstract abstract-type="short" xml:lang="es"><p><![CDATA[Las espirales contra los mosquitos se utilizan en los países de bajos ingresos como una opción para prevenir la malaria controlando el vector de esta enfermedad. A pesar de que algunos estudios han abordado este tema, se requiere más investigación para incrementar el conocimiento sobre los efectos adversos en la salud, causados por el uso prolongado de las espirales. En este estudio se investigaron los efectos toxicológicos de los gases de las espirales a partir de dos insecticidas fabricados en el país (con piretroides: transflutrina y d-aletrina como ingredientes activos) en machos de ratas albinas. Para esto, se registraron los índices hematológicos y bioquímicos, y se hicieron evaluaciones histopatológicas y de mutagenicidad en ratas expuestas a los gases de las espirales durante períodos de 2, 4, 8, 12 y 16 semanas. La determinación hematológica se realizó mediante un analizador de hematología automatizado para determinar el conteo de los Glóbulos Blancos (WBC), el Hematocrito (PCV), Glóbulos Rojos (RBC) y las Plaquetas (PLT), mientras que las evaluaciones bioquímicas se determinaron utilizando kits comerciales disponibles. Los cambios histopatológicos fuertes se estudiaron en el riñón, el hígado y los pulmones de ratas sacrificadas. Las anormalidades en la cabeza de los espermatozoides de las ratas se utilizaron para evaluar la mutagenicidad. El humo de las espirales contra los mosquitos producen un aumento significativo (p<0.05) en los niveles de proteína total, albúmina total y bilirrubina, cuando los animales fueron expuestos de dos semanas a 16 semanas con transflutrina. Del mismo modo, la elevación en las actividades de aspartato amino transferasa, alanina amino transferasa y alanina fosfatasa, aumentó significativamente con ambos insecticidas. Se registro un aumento en los leucocitos, eritrocitos y el hematocrito para todos los períodos de exposición, sin embargo el recuento de las plaquetas no mostró un aumento significativo (p&gt;0.05). Las pruebas de mutagenicidad revelaron que las anormalidades en el esperma de las ratas fue estadísticamente significativa (p&gt;0.05) al comparar el control a las 8, 12 y 16 semanas post exposición a la transflutrina. Los estudios histológicos revelaron una serie de daños pulmonares graves en las ratas expuestas al humo de la espiral, evidenciados por la acumulación intersticial, edema pulmonar y enfisema. Las acumulaciones intracelulares y la congestión sinusoidal severa de las células del hígado se observaron a partir de las 12 semanas de exposición, lo que indica daño hepático. Nuestros estudios indican que los vapores de las espirales contra mosquitos inician el daño gradual al huésped. Estos efectos patológicos deben ser tomados en cuenta por el programa de control de la malaria, particularmente a la hora de regular su uso a largo plazo y bajo techo.]]></p></abstract>
<kwd-group>
<kwd lng="en"><![CDATA[mosquito coil]]></kwd>
<kwd lng="en"><![CDATA[insecticide]]></kwd>
<kwd lng="en"><![CDATA[hematology and biochemical parameters]]></kwd>
<kwd lng="en"><![CDATA[prolong usage]]></kwd>
<kwd lng="en"><![CDATA[mutagenicity]]></kwd>
<kwd lng="en"><![CDATA[histopathology]]></kwd>
<kwd lng="es"><![CDATA[espirales contra mosquitos]]></kwd>
<kwd lng="es"><![CDATA[insecticida]]></kwd>
<kwd lng="es"><![CDATA[parámetros hematológicos y bioquímicos]]></kwd>
<kwd lng="es"><![CDATA[uso prolongado]]></kwd>
<kwd lng="es"><![CDATA[mutagenicidad]]></kwd>
<kwd lng="es"><![CDATA[histopatología]]></kwd>
</kwd-group>
</article-meta>
</front><body><![CDATA[ <div style="text-align: justify;">     <div style="text-align: center;"><font style="font-weight: bold;"  size="4"><span style="font-family: verdana;">Toxicological effects of prolonged and intense use of mosquito coil emission in rats and its implications on malaria control    <br>     <br> Efectos toxicol&oacute;gicos del uso prolongado e intenso de emisiones de espirales contra mosquitos en ratas y sus implicaciones sobre el control de la malaria<br style="font-family: verdana;"> </span></font></div> <br style="font-family: verdana;">     <div style="text-align: center;"><font size="2"><span  style="font-family: verdana;">Emmanuel Taiwo Idowu<sup><a href="#1">1</a><a  name="2"></a>*</sup>, Oyenmwen Judith Aimufua<a href="#1"><sup>1</sup></a>, Yomi-Onilude Ejovwoke<a  href="#1"><sup>1</sup></a>, Bamidele Akinsanya<a href="#1"><sup>1</sup></a> &amp; Olubumi Adetoro Otubanjo<a href="#1"><sup>1</sup></a></span></font><br  style="font-family: verdana;"> </div> <br style="font-family: verdana;"> <font size="2"><span style="font-family: verdana;">    <br>     <a name="Correspondencia2"></a>*<a href="#Correspondencia1">Direcci&oacute;n     para correspondencia:</a><br style="font-family: verdana;">     </span></font><font size="2"></font>     <hr style="width: 100%; height: 2px;"><font size="3"><span     ]]></body>
<body><![CDATA[ style="font-family: verdana; font-weight: bold;">Abstract</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">Efectos     toxicol&oacute;gicos del     uso prolongado e intenso de emisiones de espirales contra mosquitos en     ratas y sus implicaciones sobre el control de la malaria. Mosquito coil     is a vector control option used to prevent malaria in low income     counties, while some studies have addressed this issue, additional     reseach is required to increase knowledge on the adverse health effects     ]]></body>
<body><![CDATA[caused by the prolonged use of coils. In this study we investigated the     toxicological effects of fumes from two locally manufactured mosquito     coil insecticides (with pyrethroids: transfluthrin and d-allethrin as     active ingredients) on male albino rats. For this, we recorded the     haematological and biochemical indices, and made histopathology and     mutagenicity evaluations in rats exposed to mosquito fumes during 2, 4,     8, 12 and 16 week periods. Haematological determination was performed     using automated hematology analyzer to determine White Blood Cell     (WBC), Packed Cell Volume (PCV), Red Blood Cell (RBC) and Platelet     (PLT) counts, while biochemical evaluations were determined using     ]]></body>
<body><![CDATA[available commercial kits. Gross histopathological changes were studied     for the kidney, liver and lungs in sacrificed rats. The rat sperm head     abnormalities assessment was used to evaluate mutagenicity. Mosquito     coil fumes produced significant increase (P&lt;0.05) in the levels of     total protein, total albumin and bilirubin, when animals were exposed     from two weeks to 16 weeks with transfluthrin. Similarly, elevation in     the activities of aspartate amino transferase, alanine amino     transferase and alanine phosphatase, increased significantly in both     insecticides. Increase in WBC, RBC and PCV were recorded for all the     exposure periods, however PLT count showed no significant increase     ]]></body>
<body><![CDATA[(P&gt;0.05). Mutagenicity assessment revealed sperm abnormality was     statistically significant (P&lt;0.05) compared with the control at 8,     12 and 16 weeks post exposure to transfluthrin. Histological studies     revealed severe lung damage evidenced by interstitial accumulations,     pulmonary oedema and emphysema in exposed rats. Intracellular     accumulations and severe sinusoidal congestion of liver cells were     observed from 12 weeks exposure, indicating liver damage. Our studies     indicate that mosquito coil fumes do initiate gradual damage to the     host. These pathological effects must be taken into consideration by     the malaria control program, particularly when regulating their long     ]]></body>
<body><![CDATA[term and indoor usage. </span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Key words: </span>mosquito coil,     insecticide, hematology and biochemical parameters, prolong usage,     mutagenicity, histopathology.</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="3"><span style="font-family: verdana; font-weight: bold;">Resumen</span></font><br      style="font-family: verdana;">     ]]></body>
<body><![CDATA[<font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">Las espirales contra     los mosquitos     se utilizan en los pa&iacute;ses de bajos ingresos como una     opci&oacute;n para prevenir la malaria controlando el vector de esta     enfermedad. A pesar de que algunos estudios han abordado este tema, se     requiere m&aacute;s investigaci&oacute;n para incrementar el     conocimiento sobre los efectos adversos en la salud, causados por el     uso prolongado de las espirales. En este estudio se investigaron los     efectos toxicol&oacute;gicos de los gases de las espirales a partir de     ]]></body>
<body><![CDATA[dos insecticidas fabricados en el pa&iacute;s (con piretroides:     transflutrina y d-aletrina como ingredientes activos) en machos de     ratas albinas. Para esto, se registraron los &iacute;ndices     hematol&oacute;gicos y bioqu&iacute;micos, y se hicieron evaluaciones     histopatol&oacute;gicas y de mutagenicidad en ratas expuestas a los     gases de las espirales durante per&iacute;odos de 2, 4, 8, 12 y 16     semanas. La determinaci&oacute;n hematol&oacute;gica se realiz&oacute;     mediante un analizador de hematolog&iacute;a automatizado para     determinar el conteo de los Gl&oacute;bulos Blancos (WBC), el     Hematocrito (PCV), Gl&oacute;bulos Rojos (RBC) y las Plaquetas (PLT),     ]]></body>
<body><![CDATA[mientras que las evaluaciones bioqu&iacute;micas se determinaron     utilizando kits comerciales disponibles. Los cambios     histopatol&oacute;gicos fuertes se estudiaron en el     ri&ntilde;&oacute;n, el h&iacute;gado y los pulmones de ratas     sacrificadas. Las anormalidades en la cabeza de los espermatozoides de     las ratas se utilizaron para evaluar la mutagenicidad. El humo de las     espirales contra los mosquitos producen un aumento significativo     (p&lt;0.05) en los niveles de prote&iacute;na total, alb&uacute;mina     total y bilirrubina, cuando los animales fueron expuestos de dos     semanas a 16 semanas con transflutrina. Del mismo modo, la     ]]></body>
<body><![CDATA[elevaci&oacute;n en las actividades de aspartato amino transferasa,     alanina amino transferasa y alanina fosfatasa, aument&oacute;     significativamente con ambos insecticidas. Se registro un aumento en     los leucocitos, eritrocitos y el hematocrito para todos los     per&iacute;odos de exposici&oacute;n, sin embargo el recuento de las     plaquetas no mostr&oacute; un aumento significativo (p&gt;0.05). Las     pruebas de mutagenicidad revelaron que las anormalidades en el esperma     de las ratas fue estad&iacute;sticamente significativa (p&gt;0.05) al     comparar el control a las 8, 12 y 16 semanas post exposici&oacute;n a     la transflutrina. Los estudios histol&oacute;gicos revelaron una serie     ]]></body>
<body><![CDATA[de da&ntilde;os pulmonares graves en las ratas expuestas al humo de la     espiral, evidenciados por la acumulaci&oacute;n intersticial, edema     pulmonar y enfisema. Las acumulaciones intracelulares y la     congesti&oacute;n sinusoidal severa de las c&eacute;lulas del     h&iacute;gado se observaron a partir de las 12 semanas de     exposici&oacute;n, lo que indica da&ntilde;o hep&aacute;tico. Nuestros     estudios indican que los vapores de las espirales contra mosquitos     inician el da&ntilde;o gradual al hu&eacute;sped. Estos efectos     patol&oacute;gicos deben ser tomados en cuenta por el programa de     control de la malaria, particularmente a la hora de regular su uso a     ]]></body>
<body><![CDATA[largo plazo y bajo techo.</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Palabras clave:</span> espirales contra     mosquitos, insecticida, par&aacute;metros hematol&oacute;gicos y     bioqu&iacute;micos, uso prolongado, mutagenicidad,     histopatolog&iacute;a.</span></font><br style="font-family: verdana;">     <font size="2"></font>     <hr style="width: 100%; height: 2px;"><font size="2"><span      style="font-family: verdana;">Malaria is a public health problem     ]]></body>
<body><![CDATA[in many countries of the world especially in tropical and subtropical     regions. An 85% of estimated annual 225 million cases of malaria     worldwide are caused by <span style="font-style: italic;">Plasmodium     falciparum</span> and <span style="font-style: italic;">P. vivax</span>,     and are     recorded within the African region. It also accounts for approximately     one million deaths annually and 89% of the malaria death occurring in     Africa South of the Sahara (WHO 2010). In Nigeria, malaria is highly     endemic accounting for 60% of outpatient visit (Anonymous 2010a).</span></font><br      style="font-family: verdana;">     ]]></body>
<body><![CDATA[<font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">The methods employed     in the control     of malaria are solely based on the administration of antimalarial drugs     and vector control which include utilization of Insecticide Treated Net     (ITN), Long lasting Insecticide mosquito Nets (LLIN) and Indoor     Residual Spray (IRS) as recommended by the World Health Organization     (WHO). However, these options have been set back largely due to     development of resistant parasites and insecticide resistant strains of     mosquitoes (Wernsdorfer 1994, Trigg &amp; Wernsdorfer 1999, Chandre <span     ]]></body>
<body><![CDATA[ style="font-style: italic;">et     al.</span> 1999, Awolola <span style="font-style: italic;">et al.</span>     2003, Nwane <span style="font-style: italic;">et al.</span> 2009).</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">Mosquito coils     (mosquito repelling     insecticides) are essentially made up of pyrethrum powder and are     widely used in Asia, Africa and South America, particularly among the     low income earners, because of its affordability (Mulla <span     ]]></body>
<body><![CDATA[ style="font-style: italic;">et al.</span> 2001,     Weili <span style="font-style: italic;">et al.</span> 2003). The     activities of mosquito coil have been     demonstrated against <span style="font-style: italic;">Aedes</span>, <span      style="font-style: italic;">Anopheles </span>and <span      style="font-style: italic;">Mansonia</span> (Yap <span      style="font-style: italic;">et al.</span> 1996,     Lawrence &amp; Croft 2004). The health implications of burning one     mosquito coil is equivalent to the release of the same amount of     particulate matter as burning 75 to 137 cigarettes, and emitting     ]]></body>
<body><![CDATA[formaldehyde equivalent to 51 cigarettes (Chen <span      style="font-style: italic;">et al.</span> 2008, Liu <span      style="font-style: italic;">et al.</span>     2003).</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">Histopathological     investigations     have demonstrated that mosquito fumes leads to focal dociliation of the     tracheal epithelium, metaplasia of the epithelial cells and     morphological alteration of the alveolar macrophages (Liu &amp; Sun     ]]></body>
<body><![CDATA[1988, Liu &amp; Wong 1987, Cheng <span style="font-style: italic;">et     al.</span> 1992, Chang &amp; Lin 1998).     Kidney tissues of exposed rats have revealed severe multifocal     congestion, cystic dilation in the medulla, interstitial mononuclear     cellular infiltration and wide spread fibrosis (Garba <span      style="font-style: italic;">et al.</span> 2007a,     Taiwo <span style="font-style: italic;">et al.</span> 2008), while     damage to spleen revealed severe sinusoids     hyperplasia and regression of red and white pulps (Garba <span      style="font-style: italic;">et al.</span> 2007b).</span></font><br     ]]></body>
<body><![CDATA[ style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">Elevated levels of     urea and     creatinine have been reported in rats exposed to mosquito smoke (Garba     <span style="font-style: italic;">et al.</span> 2007a) and significant     increase in white blood cell count Garba     <span style="font-style: italic;">et al.</span> 2007b). Mutagenicity     effect of mosquito coil smoke have been     reported to cause chromosomal aberrations in metaphases and a     ]]></body>
<body><![CDATA[significantly higher incidence of chromosomal aberration frequency in     exposed rats and mice (Das <span style="font-style: italic;">et al.</span>     1994, Moorthy &amp; Murthy 1994).     Besides, long term exposure to mosquito coil smoke has been     demonstrated by some workers to induce asthma and persistent sneezing     in children (Azizi &amp; Henry 1991, Fagbule &amp; Ekanem 1994, Koo     &amp; Ho 1994).</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">Presently, the use     of mosquito coil     ]]></body>
<body><![CDATA[as a control option is widespread in Nigeria, despite the fact that     inhaling mosquito coil fumes may have potential adverse health effects.     Here we investigated the histopathological alteration on various     organs, the changes in haematological indices and the mutagenicity     induced in mice with a model of prolonged use of coil.</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font style="font-weight: bold;" size="3"><span      style="font-family: verdana;">Materials and Methods</span></font><br      style="font-family: verdana;">     ]]></body>
<body><![CDATA[<font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Tests materials:</span> Two different     mosquito coils were sourced from Nigerian markets and were used for the     experiment namely Baygon and Raid. Baygon brand (containing 0.03%     transfluthrin and 99.7%w/w inert ingredients) manufactured by Turare     N&#8217;Hausawa Ltd. In Kano State. Raid brand (containing pyrethroids     (d-allethrin) 0.2%w/w and other ingredients 99.8%) manufactured by     Johnson Wax Nigeria Ltd, Isolo, Lagos State.</span></font><br      style="font-family: verdana;">     ]]></body>
<body><![CDATA[<font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Test animals:</span> A total of 60 albino     rats (<span style="font-style: italic;">Rattus rattus norwegicus</span>),     all adult males weighing     210&plusmn;30g were used for this study. The rats were purchased from     the Animal Facility Centre of Biochemistry Department, Nigerian     Institute of Medical Research, Yaba, Lagos. The rats were kept in the     Zoological Garden of the University of Lagos for two weeks to     acclimatize to their new environment, and were observed for physical     ]]></body>
<body><![CDATA[deformity or any ailment that may render them unfit for the study. The     exposure to the insecticide coil, namely Baygon and Raid brands, was     performed in two different rooms, which were far apart such that the     emission driven in one room did not affect the other. The animals were     kept in locally fabricated cages with a size of 30x60cm=180sqcm<sup>2</sup>.     The     cages were made of plastic at the base and sides, and the dimension of     wire mesh used to cover the cages were 0.5x0.5cm (1/4&#8221;x1/4&#8221;) square     openings. The animals were kept at a room temperature of 30&plusmn;3oC     and a relative humidity of about 35% with a 12hr light/dark cycle. They     ]]></body>
<body><![CDATA[had access to drinking water and standard laboratory diet <span      style="font-style: italic;">ad libitum</span>.</span></font><br      style="font-family: verdana;">     <font size="2"></font><br      style="font-family: verdana; font-weight: bold;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Experimental design:</span> For the     exposure of two different mosquito coils (transfluthrin and     d-allethrin), the rats were divided into six groups. The rats were kept     in different plastic boxes of four rats each. The rats in groups one to     ]]></body>
<body><![CDATA[five were exposed, while those in group six were unexposed and served     as the control group. The rats in group one were exposed for two weeks,     group two for four weeks, group three for eight weeks, group four for     twelve weeks, and group five for 16 weeks; after exposure, all rats     were slaughtered. The rats in group 6 were not exposed to the fumes at     all; each time rats from any group were slaughtered, two rats from the     control group were also slaughtered for comparison. The rats in groups     one through five were kept in a separate location from the control, to     prevent accidental or low level exposure.</span></font><br      style="font-family: verdana;">     ]]></body>
<body><![CDATA[<font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">The cages were     demarcated with wire     mesh to allow a small space for the mosquito coil which was in a     separate compartment from the rats to prevent the rats from knocking     over the coil and starting a fire, but allowing the smoke to penetrate     and saturate the box allowing maximum exposure of the rats to the     fumes. The box was covered round about and a wire mesh on a window cut     on top of the box to allow for ventilation. They were exposed for eight     hours a day for the period of study, as an attempt to mimic average     ]]></body>
<body><![CDATA[period of time that man sleeps in a day (Anonymous 2012b).</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">The rats in each     group were     observed for any clinical signs associated with the exposure to the     active ingredient from the fumes. The rats were killed using the method     of spinal paralysis and dissected to obtain the kidneys, lungs and     liver for histopathological studies while 3mL of blood samples were     obtained for determination of haematological and quantitative     ]]></body>
<body><![CDATA[determination of total Protein (PR), Albumin (ABL), Alanine     aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline     Phosphatase (ALP), Serum Bilirubin (BIL) and blood Urea nitrogen (UR).</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Haematological determination:</span> Blood     samples were collected in appropriate heparinized blood containers for     determination of the Packed Cell Volume (PCV), Red Blood Cell (RBC),     White Blood Cell (RBC) using automated hematology analyzer (Minray     ]]></body>
<body><![CDATA[Model BC 3200).</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Biochemical analysis: </span>The     biochemical assessment was done with rat blood samples. Alanine     aminotransferase level was measured by commercially available standard     blood ALT kit by Randox Reitman and Frankel ALT level 2 control (cat.     No. SC 2643). The method of Young (1975), was used to determine     alkaline phosphatase (ALP). The BCG method of Doumas <span      style="font-style: italic;">et al.</span> (1985) was     ]]></body>
<body><![CDATA[used to estimate Albumin. The plasma urea level was measured by     commercially available standard blood urea kit (Maizy, France) by     Biolabo South Africa. The plasma creatinine level was measured by     commercially available standard blood creatinine kit (Maizy, France) by     standard protocol for photometric determination of creatinine based on     Jaffe kinetic method. The biuret method was used for the measurement of     protein level. The Sulfanilic acid method of Tietz &amp; Shuey (1993)     was employed for the estimation of bilirubin.</span></font><br      style="font-family: verdana;">     <font size="2"></font><br     ]]></body>
<body><![CDATA[ style="font-family: verdana; font-weight: bold;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Mutagenicity determination:</span>     Mutagenicity was determined by examination of the sperm head     abnormalities after testicles dissections that followed the methods of     Wyrobek &amp; Bruce (1975). The control and experimental groups were in     replicates (A and B). A thin smear of the spermatozoa was made on a     slide and fixed with 95% ethanol and allowed to air dry for about     5-10min. The smear was washed with sodium bicarbonate formalin solution     to remove any mucus that may be present. The slide was rinsed several     ]]></body>
<body><![CDATA[times in water and the smear covered with dilute carbon fuels in     solution (1:20), and allowed to stain for about 3min, washed off with     water subsequently stained again with dilute Loeffer&#8217;s methylene blue.     The slides were then coded, randomized and examined cytologically under     X100 oil immersion binocular light microscopy. Three separate slides     were prepared for each group out of which two slides were selected for     scoring using the tally counter. For each group, 800 sperm cells were     assessed for morphological aberration according to the criteria of     Wyrobek &amp; Bruce (1975). Mutagenicity assessment was only carried in     animals exposed to d-allethrin.</span></font><br     ]]></body>
<body><![CDATA[ style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">All data from this     study were     expressed as mean&plusmn;standard error. A probability level of less     than 5% (p&lt;0.05) was considered significant in all instances.     Statistical software version 5.03 (1992-2010).</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="3"><span style="font-family: verdana; font-weight: bold;">Results</span></font><br     ]]></body>
<body><![CDATA[ style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Haematological indices:</span> The effects     of mosquito coil smoke inhalation on haematological indices are     presented in <a href="/img/revistas/rbt/v61n3/a36t1.gif">table 1</a>.     RBC and PCV increased in all groups exposed to     mosquito coil smoke and was observed to be significant (p&lt;0.05) in     the groups of rats exposed for two and eight weeks and not significant     (p&gt;0.05) for 12 and 16 weeks when compared with the control for rats     ]]></body>
<body><![CDATA[exposed to transfluthrin. However rats exposed to d-allethrin were     observed not to be significant (p&gt;0.05) for two and four weeks but     significant (p&lt;0.05) as from eight to 16 weeks when compared with     control.</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">Platelets counts     showed no     significant change (p&gt;0.05) for various experimental groups when     exposed rats were compared to non-exposed rats for both transfluthrin     and d-allethrin.</span></font><br style="font-family: verdana;">     ]]></body>
<body><![CDATA[<font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">WBC counts increased     in all the     exposed groups when compared with control groups, and was not     significant for all groups of rats exposed to transfluthrin     (p&gt;0.05), but significant (p&lt;0.05) for all groups exposed to     d-allethrin from four to 12 weeks.</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span     ]]></body>
<body><![CDATA[ style="font-weight: bold;">Biochemical assessments:</span> <a      href="/img/revistas/rbt/v61n3/a36t2.gif">Table 2</a>     presents some biochemical assessment of mosquito coil emission in     albino rats. AST, ALP and ALT levels increased significantly in all     animals groups exposed (two to 16 weeks) to mosquito coil smoke     (p&lt;0.05) when compared with the control animals for both     transfluthrin and d-allethrin.</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">Significant levels     ]]></body>
<body><![CDATA[(p&lt;0.05) of     total protein, albumin and creatinine were recorded for rats exposed to     transfluthrin when all experimental groups were compared with control     animals. Serum levels of creatinine, albumin and total protein were not     significantly affected for animals exposed to d-allethrin.</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">Blood urea levels     were not     significant for all groups of rats exposed to d-allethrin and two and     ]]></body>
<body><![CDATA[four weeks exposed to transfluthrin but significant for eight and 16     weeks when they were compared with the control. Bilirubin level were     increased significantly (p&lt;0.05) in the groups of rats exposed to     transfluthrin mosquito coil smoke for four and 16 weeks, and 12 and 16     weeks for rats exposed to d-allethrin.</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Histopathological findings:</span> The     histopathology for the two mosquito coil (transfluthrin and     ]]></body>
<body><![CDATA[d-allethrin) used followed the same pattern for the organs investigated     namely kidney, liver and lungs.</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Kidney: </span>Kidney tissue of albino     rats exposed to mosquito coil smoke showed no morphological changes     until eight weeks, when mild interstitial congestion were first     observed, however, full congestion around the glomerular tuft were     observed in the 16 weeks post exposure (<a     ]]></body>
<body><![CDATA[ href="/img/revistas/rbt/v61n3/a36i1.jpg">Fig. 1A</a>). <a      href="/img/revistas/rbt/v61n3/a36i1.jpg">Figure 1B</a> evidenced     the control albino rats showing normocellular glomerular tufts in a     background of tubules with cuboidal cell epithelial lining with no     necrosis.</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Liver: </span>Histological appearance of     liver tissues exposed to mosquito coil smoke for two weeks showed     extensive intracytoplasmic accumulations and moderate hydropic change.     ]]></body>
<body><![CDATA[The liver tissues of rats exposed for four weeks showed generalized     intracellular accumulations, and the cytoplasm appeared frosted and     granular; there was also a mild hydropic change. At eight weeks of     exposure, the liver tissues showed preserved architecture hepatocytes     displayed as radiating plates and uniformly eosinophilic cytoplasm,     while liver tissues of experimental animals exposed for 12 and 16     weeks, showed generalized intracellular accumulations and severe     sinusoidal congestion (<a href="/img/revistas/rbt/v61n3/a36i1.jpg">Fig.     1C</a>). Histological appearance of liver     tissue of control animals showed normal hepatocytes, arranged as     ]]></body>
<body><![CDATA[plates, nucleus surrounded by a nuclear membrane and nucleolus (<a      href="/img/revistas/rbt/v61n3/a36i1.jpg">Fig.     1D</a>).</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Lungs:</span> Histological appearance of     lung tissues of animals exposed to mosquito coil smoke for two weeks     showed mixed inflammatory cells, giant cell reaction, stromal fibrosis,     and four weeks group showed inflammation and congestion of the     interstitium, and hyperplasia of peribronchial lymphoid aggregates. At     ]]></body>
<body><![CDATA[16 weeks of exposure, the lung tissues showed peribronchial lymphoid     hyperplasia, interstitial inflammation and congestion with pulmonary     oedema (<a href="/img/revistas/rbt/v61n3/a36i1.jpg">Fig. 1E</a>). <a      href="/img/revistas/rbt/v61n3/a36i1.jpg">Fig. 1F</a> shows     histological appearance of control lung     tissue.</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;"><span      style="font-weight: bold;">Mutagenicity assessment:</span> Nine     different forms of sperm head abnormality were observed from sperm     ]]></body>
<body><![CDATA[squashed in the sperm head abnormality assay. The abnormal sperm showed     rudimentary tail, bent mid-piece, curved mid-piece, bent tail, curved     tail, normal head without tail/tailless head, normal tail without     head/headless tail, coiled tail and looped tail. <a      href="/img/revistas/rbt/v61n3/a36t3.gif">Table 3</a> shows the mean     and percentage of normal sperm cells and abnormal sperm cells of     control and experimental animals. There was no statistical difference     in the percentage of sperm head abnormality of animals exposed to     mosquito coil smoke during two and four weeks (p&gt;0.05), when     compared with that of the control, however for eight, 12 and 16 weeks     ]]></body>
<body><![CDATA[post exposure, it was resulted highly significant (p&lt;0.05).</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="3"><span style="font-family: verdana; font-weight: bold;">Discussion</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">The significant     increase in RBC and     PCV in rats exposed to transfluthrin at two and four weeks and     d-alletrin at two to 12 weeks, may be due to cyanide which is a by     ]]></body>
<body><![CDATA[product of mosquito coil smoke which is known to cause reduction in     oxygen carrying capacity of RBC leading to reduced metabolism. The     reduction of oxygen stimulates erythropoietin which in turn stimulates     the bone marrow to produce RBC. These findings are in agreement with     earlier workers (Parker <span style="font-style: italic;">et al.</span>     1984, Schoeinig 1995, Shakouri <span style="font-style: italic;">et al.</span>     1992, Garba <span style="font-style: italic;">et al.</span> 2007a)     which similarly observed increase in RBC and     PCV in rats exposed to pyrethrins however it is contrary to work of     Saka <span style="font-style: italic;">et al.</span> (2011) which     ]]></body>
<body><![CDATA[demonstrated no significant increase in     haematological parameters (WBC, RBC and PLT) when they exposed rats for     one to three daily hours; however, the present study exposed the rats     for eight hours daily mimicking daily exposure of man to mosquito coil     in normal setting. Since the study further reveals the RBC and PCV     levels dropped and became insignificant compared with control at 16     weeks exposure to both transfluthrin and d-allethrin insecticide, it     can be concluded that haematological parameters observed do not suggest     anemia as a risk factor in mosquito coil exposure. However marked     increase in WBC recorded in rats exposed to d-allethrin in this study     ]]></body>
<body><![CDATA[confirm observation of Garba <span style="font-style: italic;">et al.</span>     (2007a).</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">The elevated     activities of AST, ALT     ALP enzymes observed in rats exposed to both transfluthrin and     d-allethrin insecticides suggest hepatic damage or dysfunction; high     concentrations of these enzymes in hepatic tissues of dogs, cats and     primates have been linked to hepatocellular damage (Kaneko &amp;     Cornelius 1980, Hall <span style="font-style: italic;">et al.</span>     ]]></body>
<body><![CDATA[2001). This was confirmed by     histopathological examination of rats exposed to transfluthrin and     d-allethrin mosquito coil smoke for eight, 12 and 16 weeks in this     study, which indicates generalized intracellular accumulations and     severe sinusoidal congestion, which also have been demonstrated (Garba     <span style="font-style: italic;">et al.</span> 2007b, Taiwo <span      style="font-style: italic;">et al.</span> 2008, Aslam <span      style="font-style: italic;">et al.</span> 2010). This study     therefore demonstrates potential damage to the liver arising from the     use of transfluthrin and d-allethrin mosquito coil. Fetoui <span     ]]></body>
<body><![CDATA[ style="font-style: italic;">et al.</span>     (2010) demonstrated increase in the enzymatic activities of     aminotransferases AST and ALT when exposed to Lambda-Cyhalothrin     (pyrethroid) which is ameliorated with co-administration of vitamin C,     similarly Cypermethrin, a synthetic pyrethroid insecticide, have been     shown to increase liver enzymes levels of broiler chicks which were     ameliorated by combination of Vitamin E and selenium (Aslam <span      style="font-style: italic;">et al.</span>     2010). It is therefore suggested that the administration of Vitamins C     and E, may reduce liver damage and may be recommended as routine     ]]></body>
<body><![CDATA[intakes for those using pyerthroid based insecticide for mosquito     control.</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">The inflammation     resulting from     damage to the liver cells, which are sites of the protein synthesis,     may lead to the increase level of plasma protein. The coil smoke could     lead to elevated plasma urea levels; the elevation could probably be     due to the increase in activities of urea enzymes, ornithine carbomoyl     transferase and arginase, which provide evidence of liver damage in     ]]></body>
<body><![CDATA[many animal species. The concentration of total protein, total albumin     and creatinine which are a measure of liver and kidney function, are     significantly higher when compared to the control groups in rats     exposed to transfluthrin, while the reverse is the case in animal     exposed to d-alletrin. Exposure to transfluthrin also demonstrated     significant levels of bilirubin and blood urea in some exposed groups     which agrees with the findings of (Liu <span      style="font-style: italic;">et al.</span> 1989, Garba <span      style="font-style: italic;">et al.</span> 2007b)     the observation of this study therefore suggest that transfluthrin can     ]]></body>
<body><![CDATA[cause more damage to the liver and kidney than raid but these     differences is not noticeable in histological findings.</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">Histopathological     evaluation of     mosquito coil effect had shown the impact on the kidney, 16 weeks post     exposure, which demonstrates full congestion around the glomerular     tuft, the study agrees with Taiwo <span style="font-style: italic;">et     al.</span> 2008 which demonstrated     ]]></body>
<body><![CDATA[glomerula and tubular degeneration, necrosis, thrombosis and vasculitis     to mosquito coil and varying insecticidal spray fumes in experimental     rats. The study is also in line with earlier published work of Garba <span      style="font-style: italic;">et     al.</span> 2007a which demonstrated serve multifocal congestion, cystic     dilation in the medulla of kidney tissue exposed to pyrethroid based     mosquito coil.</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">The lung tissue of     rats exposed to     ]]></body>
<body><![CDATA[mosquito coil smoke for the longest period (16 weeks) showed     peribronchial lymphoid hyperplasia, interstitial and congestion     pulmonary oedema; this observation has been documented by Okine <span      style="font-style: italic;">et al.</span>     2004. Oedema could have resulted from the inflammatory processes taking     place as a result of irritation of various organs by toxic chemicals     from coil smoke. Other pathological manifestation that has been     associated with pyrethroid mosquito coil but not observed in this     study, includes exudative pneumonia, anthracosis, thrombosis and     vasculitis, as observed by Taiwo <span style="font-style: italic;">et     ]]></body>
<body><![CDATA[al.</span> 2008. The sneezing that     resulted at the initial stage of exposure could be the result of     irritants released in the coils smoke such as aldehydes, sulphates and     polycyclic aromatic hydrocarbons such as acenaphthene, penanthrene,     benzo(a)pyrene (EPA 1998, Liu <span style="font-style: italic;">et al.</span>     2003).</span></font><br style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">The present study     demonstrates that     mutagenicity was induced and was statistically significant as from     ]]></body>
<body><![CDATA[eight weeks exposure to mosquito coil smoke. The abnormal spermatozoa     can therefore cause significantly reduction in the reproductive     potential of the male rats. High frequency of chromosomal aberration in     metaphase have been reported in rats and mice exposed to mosquito coil     (Das <span style="font-style: italic;">et al.</span> 1994, Moorthy     &amp; Murthy 1994). The potential of     induction of mutagenicity of mosquito coil in humans is therefore a     major concern, because it could account for infertility in males, as     was also demonstrated by Madhubabu &amp; Yenugu (2012) in a study of     the effect of continuous inhalation of allethrin based mosquito smoke     ]]></body>
<body><![CDATA[in the male reproductive tract of rats. The findings of this     preliminary studies therefore demands further investigation on     experimental mice and human.</span></font><br      style="font-family: verdana;">     <font size="2"></font><br style="font-family: verdana;">     <font size="2"><span style="font-family: verdana;">Malaria control     heavily rely on the     use of pyrethroids, however the use of pyrethroids in form of mosquito     coil is an available option especially to the rural populace. Control     effort aiming at reducing man contact with the smoke should be     ]]></body>
<body><![CDATA[encouraged in view of histopathological damage and the mutagenicity it     can produce due to long term exposure.</span></font><br      style="font-family: verdana;">     <font size="2"></font>     <hr style="width: 100%; height: 2px;"><font size="3"><span      style="font-family: verdana; font-weight: bold;">References</span></font><br      style="font-family: verdana;">     <br style="font-family: verdana;">     <!-- ref --><div style="text-align: left;"><font size="2"><span  style="font-family: verdana;">Anonymous. 2012a. National Malaria Control Programme. Federal Ministry of Health, Abuja, Nigeria. (Downloaded: February 28, 2012, www.nmcpnigeria.org/).    &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;[&#160;<a href="javascript:void(0);" onclick="javascript: window.open('/scielo.php?script=sci_nlinks&ref=1656181&pid=S0034-7744201300040003600001&lng=','','width=640,height=500,resizable=yes,scrollbars=1,menubar=yes,');">Links</a>&#160;]<!-- end-ref --><!-- ref --></span></font><br  style="font-family: verdana;"> <br style="font-family: verdana;"> <font size="2"><span style="font-family: verdana;">Anonymous. 2012b. American Time Use Survey. U.S. Bureau of Labor Statistics (USBLS). Washington, D.C., USA. 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Emmanuel Taiwo Idowu</span></small><font size="2"><span  style="font-family: verdana;"><small>. </small>Department of Zoology, University of Lagos, Akoka, Lagos, Nigeria; etidowu@yahoo.com    <br> </span></font><font size="2"><span style="font-family: verdana;">Oyenmwen Judith Aimufua</span></font><font size="2"><span  style="font-family: verdana;">. Department of Zoology, University of Lagos, Akoka, Lagos, Nigeria; judi4u24@yahoo.com    <br> </span></font><font size="2"><span style="font-family: verdana;">Yomi-Onilude Ejovwoke</span></font><font size="2"><span style="font-family: verdana;">. Department of Zoology, University of Lagos, Akoka, Lagos, Nigeria; ejovwokea@yahoo.co.uk    <br> </span></font><small><span style="font-family: verdana;">Bamidele Akinsanya</span></small><font size="2"><span  style="font-family: verdana;">. Department of Zoology, University of Lagos, Akoka, Lagos, Nigeria; akinbami200@yahoo.com    <br> </span></font><font size="2"><span style="font-family: verdana;">Olubumi Adetoro Otubanjo</span></font><font size="2"><span  style="font-family: verdana;">. Department of Zoology, University of Lagos, Akoka, Lagos, Nigeria;&nbsp; adetoro2001@yahoo.com</span></font><br style="font-family: verdana;"> <font size="2"></font></div> <hr style="width: 100%; height: 2px;">     <div style="text-align: center;"><font size="2"><span  style="font-family: verdana; font-weight: bold;">Received 04-VI-2012. Corrected 12-XII-2012. Accepted 22-I-2013.</span></font><br style="font-family: verdana;"> </div> <font size="2"> </font>      ]]></body><back>
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