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    C 3: C

    3.1. B

    Ctotoicit testing is one of the major assas applied dring toin assessment,

    hich focses mainl on cell death or some measre of groth impairment. This tpe of

    testing is designed to ealate the intrinsic abilit of a compond to kill cells (Ferro & Dole,

    2001). Apart from dosage, to other factors pla a major role in the toicolog of an entit:

    the dration of eposre to a compond and the componds mechanism of toicit (Riss &

    Moraec, 2004). At a celllar leel, toicit can manifest in a nmber of as

    inclding (Horath, 1980):

    1. diminished celllar adhesion

    2. dramatic morphological changes

    3. a decrease in replication rate or

    4. a redction in oerall iabilit

    Man different assas hae been deeloped to determine toicit sch as

    qantifing cell death/srial b assessing plasma membrane integrit, cell enmeration b

    total protein content and enmerating iable cells throgh assessing certain ital fnctions.

    Poplar assas that are idel sed are the total celllar protein assa (slforhodamine B),

    the netralred ptake assa, the LDH leakage assa, and the tetraolim de assas

    (Mrakami, 2000).

    The best knon of the tetraolim assas is probabl the MTT assa for mammalian cells in

    hich 3(4,5Dimethlthiaol2l)2,5diphenltetraolim bromide, a ello tetraolim

    salt is redced b the mitochondria of iable cells to insolble, prple formaan crstals.

    Althogh this is one of the most prealent iabilit assas, its eakness lies in ielding false

    posities in the presence of a nmber of componds inclding albmin and antioidants

    sch as ascorbic acid, csteine and gltathione (Fnk ., 2007).

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    The netral red ptake (NRU) assa is also idel sed to determine cell iabilit. Netral

    red, a spraital (nontoic) de, relies on the principle of de accmlation in the

    lsosomes and Golgi apparats of iable, ninjred cells and has the folloing adantages:

    1. it does not rel on a redction reaction to determine iabilit, eclding

    ssceptibilit to making tpe I errors (false posities) in the presence of antioidants

    or other redctie agents

    2. it is costeffectie

    3. it is qick

    4. is reported to be more sensitie to changes in cell iabilit of hepatocte cltres as

    compared to total protein content determination and the LDH leakage assa (Fotakis

    and Timbrell, 2006)

    In this chapter the effects of DDT, DDE and DDD on hepatocte iabilit are presented. Initial

    ctotoicit testing as performed in order to establish a concentration range that old be

    sed throghot the std. The concentration range that as selected coered a ide

    toicit range, from merel affecting cell fnction to complete loss of celllar fnction and

    iabilit. This as done in order to assess the incremental effects of the test componds on

    different parameters at different leels of toicit, hich ma shed light on the optimal

    concentration range that can be tilised ith the procedre deeloped in the present std.

    3.2.

    3.2.1. E

    After a 48 h incbation period (to allo cells to adhere to the ells), cells ere eposed to

    DDT, DDE and DDD at concentrations of 5, 10, 50, 100 and 150 M. Tamoifen (150M),

    hich is knon to indce cell death throgh apoptosis in HepG2 cells (Go ., 2010), as

    sed as positie control. DMSO (0.5%) as inclded as ehicle control. After eposre, cells

    ere incbated for 24 h at 37C ntil performing the iabilit assa. To assess the possible

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    hepatoprotectie effects of NAC, three additional eperiments ere condcted, hich

    inclded an additional 1 h pretreatment ith 620 nM NAC, prior to test compond

    eposre.

    3.2.2.

    Eposre medim as aspirated and replaced ith 100 l EMEM containing 100 g/ml

    Netral red de. Cells ere incbated ith the de for 2 h at 37C, after hich medim as

    discarded and cells ere ashed ith PBS (200 l). Plates ere dried oernight at 40C and

    the accmlated de dissoled b adding 100 l of Netral red eltion bffer to each ell

    and incbated at room temperatre on an orbital shaker for 40 min (Fotakis and Timbrell,

    2006). The amont of de accmlated b the cells in each ell as qantified b

    measring the absorbance at 540 nm ith a reference aelength of 630 nm (Biotek XL

    plate reader).

    3.2.3.

    Si eperiments ere carried ot in dplicate ( 12) to assess iabilit response. All

    obsered reslts ere standardised to percentage of control ales. Qalit of the collected

    data as assessed b plotting the distribtions for the tested concentration of each tested

    compond. Grbb's test as performed to remoe otliers, after hich a ShapiroFrancia

    test as sed to assess normalit based on a discrete nmber. Depending on the normalit

    of the data, stdent's tests (parametric) or MannWhitne tests (nonparametric) ere

    performed across the respectie concentration ranges to determine hether the different

    concentrations had an statisticall significant inflence on HepG2 iabilit. Three additional

    eperiments ere carried ot in dplicate to assess the possible effects that NAC ma hae

    on the initiall obsered toicit. These reslts ere also standardised to percentage of

    control bt no preliminar tests (Grbb's and ShapiroFrancia) ere performed. De to the

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    small sample sie ( 6), no otliers ere remoed and normalit of the data cold not be

    established. Therefore, all NAC reslts ere analsed sing MannWhitne tests. The 50%

    Inhibitor concentrations (IC50) ere determined b fitting a Hill eqation ith ariable

    slope to the obsered data. Analses ere performed sing GraphPad Prism 5.0

    (.graphpad.com) and the freeare package, R 2.12.1 (.rproject.org). All reported

    reslts are gien as mean standard error of the mean (SEM), nless stated otherise.

    3.3.

    Normalit testing reealed nonnormal distribtions, especiall for the higher

    concentrations of the tested componds. The different distribtions for DDT, DDE and DDD

    are illstrated in Figres 3.1, 3.2 and 3.3, respectiel. Nonnormalit of some distribtions

    as confirmed ith the ShapiroFrancia test (Table 3.1). The ShapiroFrancia test is based

    on hpothesis testing: the nll hpothesis states that the collected data originates from a

    normal distribtion, if theale drops belo the chosen alpha leel, the nll hpothesis is

    rejected and the data assmed to originate from a nonnormal distribtion. Concentrations

    beteen 50 M and 150 M for DDT and DDD and 100 M for DDE ere fond to be

    significantl nonnormal. The Control grop in the DDT data set is an eample of a perfectl

    normall distribted data set ith= 1.00 (Figre 3.1A).

    DDT did not hae an significant inflence on iabilit p to concentrations of 10 M bt did

    prodce slight celllar proliferation ith enmeration reslts being 105.4 3.5% and 103.4

    3.8% for concentrations of 5 M and 10 M, respectiel (Figre 3.4). Higher

    concentrations reslted in significant ( < 0.001) decreases in iabilit. Concentrations of 50,

    100 and 150 M DDT indced 54.3 3.7%, 74.5 4.8% and 74.4 3.8% losses in iabilit,

    respectiel (Figre 3.4). The Hill eqation fitted the obsered DDT reslts ell ith a

    coefficient of determination () of 0.86, meaning that 86% of the ariance obsered in the

    reslts can be acconted for b the fitted model (Figre 3.4). From the fitted eqation it as

    dedced that DDT has an IC50 ale of 54 1 M after 24 h eposre, if a cell densit of

    2104cells/ell is sed (Table 3.2).

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    NAC pretreatment did not alleiate bt rather aggraated DDT toicit, decreasing the IC 50

    to 40 1 M, after 24 h eposre (Table 3.2 and Figre 3.4). Compared to DDT eposre

    alone, NAC pretreatment significantl decreased the iabilit at lo concentrations of 5 M

    ( < 0.05) and 10 M ( < 0.01) (Figre 3.4).

    Figure 3.1. Histogram density plots of the observed viability data of HepG2 cells exposed to DDT

    demonstrating the distributions of the collected data. The control group is a good example of a normal

    distribution. The observations did not always follow a normal distribution, especially in the higher ranges

    of viability. X-axis represents observed values and Y-axis, the count.

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    Figure 3.2. Histogram density plots of the observed viability data of HepG2 cells exposed to DDE

    demonstrating the distributions of the collected data. The results do not follow a normal distribution. X-

    axis represents observed values and Y-axis, the count.

    Figure 3.3. Histogram density plots of the observed viability data of HepG2 cells exposed to DDD

    demonstrating the distributions of the collected data. Data is non-normal as indicated with distinct

    multiple peaks instead of a single peak. X-axis represents observed values and Y-axis, the count.

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    Similar to the parent molecle, DDE prodced a dosedependent decrease in iabilit.

    Concentrations p to 10 M did not prodce an significant flctations in iabilit.

    Hoeer, nlike DDT, it cased a slight decrease in iabilit at the 5 M and 10 M

    concentrations, ielding 97.4 1.5% and 97.5 2.5% iabilit, respectiel (Figre 3.4).

    Concentrations beteen 50 M and 150M prodced significant deiation from the control

    mean ( < 0.001). A 50 M concentration of DDE cased a 37.1 2.0% decrease in iabilit,

    hile 100 and 150 M