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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 loered cell iabilit b 73.1 3.3% and 82.6 2.0% respectiel

(Figre 3.4). At a cell densit of 2104cells/ell, DDE demonstrated an IC50of 64 1 M (

= 0.95) after 24 h eposre (Table 3.2 and Figre 3.4).

Again, NAC pretreatment did not alleiate the toicit of the test compond, decreasing the

IC50to 58 1 M, after 24 h eposre (Table 3.2 and Figre 3.4). When compared to DDE

eposre alone, NAC pretreatment significantl ( < 0.05) decreased the iabilit at

concentrations of 10 M and 50 M (Figre 3.4).

Table 3.1. Shapiro-Francia test results for normality of the observed data. Values given in

the table are p-values and instances where p < 0.05 are not normally distributed (*).

C DD DDE DDD

C 1.00 0.91 0.54

5 0.22 0.39 0.23

10 0.33 0.12 0.64

50 0.02* 0.33 0.02*

100 0.02* 0.03* 0.01*

150 0.02* 0.24 0.01*


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Table 3.2. IC50 values (SEM) of cells with/without NAC pre-treatment prior to test

compound exposure.

() AC ()

DD 54 1 40 1

DDE 64 1 58 1

DDD 44 1 33 1

Similar to DDT and DDE, DDD also prodced a dosedependent decrease in HepG2 iabilit.

DDD did not significantl affect HepG2 iabilit p to concentrations of 10 M, ith 5 M

and 10 M concentrations ielding iabilities of 99.1 2.1% and 99.0 2.3%, respectiel.

Hoeer, concentrations of 50 M indced significant ( < 0.001) decreases in iabilit

(Figre 3.4). The Hill eqation fitted the obsered data ell (= 0.91). From this analsis,

DDD shoed an IC50of 44 1 M after 24 h eposre (Figre 3.4).

NAC pretreatment prodced reslts similar to those obsered ith DDT and DDE b not

alleiating the toicit of the test compond at lo test compond concentrations. The IC50

as decreased to 33 1 M, after 24 h eposre (Table 3.2 and Figre 3.4). Compared to

DDD eposre alone, NAC pretreatment significantl ( < 0.001) decreased the iabilit at a

concentration of 50 M (Figre 3.4).


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Figure 3.4. Fitted dose-response curves of viability of HepG2 cells after DDT, DDE and DDD

treatment (mean SEM). Dark green curves represent the test compounds alone and light green curves,

cells pre-treated with NAC. Curves were obtained by fitting viability results to a four-parameter Hill

equation with variable slope and the following constraints: top = 100 and bottom = 0. Graphs are plotted

on semi-logarithmic axes. Dashed horizontal lines represents Y = 50%. #### = pppp< 0.001, treatment with test

compound alone compared to controls. $$$$ = pppp< 0.05, $$$$$$$$ = pppp< 0.01, $$$$$$$$$$$$ = pppp< 0.001, pre-treatment with

NAC compared to treatment with test compound alone.


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3.4. D

Figres 3.1 3.3 present the data distribtion plots of the obsered data for the different

concentrations of DDT, DDE and DDD, respectiel. In an ideal sitation these cres old

resemble a bellshaped cre. Rather than describing the endpoint, these cres are good

for describing the obsered data itself and proide gidance for sbseqent statistical

maniplation. Noticeable from the graphs of some of the data are more than one peak. This

is indicatie of more than one poplation being present in a particlar set of data. For

eample, these cres are er closel related to histograms obtained from flo ctometr.

If the Xais of a histogram represents sie and the Yais cont or densit, a mitre of

ooctes and spermatooa ill present as to distinct poplations in terms of sie. In the

same a, the cres presented here sho the nmber of obserations made at a particlar

ale, hich in this case old be a % of control ale. Hoeer, nlike flo ctometr,

increasing the nmber of repetitions old eentall prodce a perfect bellshaped cre.

More than one poplation indicates ariabilit beteen eperiments. In this a these

cres can be interpreted as a measre of the robstness of an assa. Hoeer, care shold

be taken hen interpreting these cres as mltiple poplations ma also be the reslt of

otliers. For this reason, otliers identified b the Grbbs test ere remoed prior to

testing for normalit (ShapiroFrancia test). Data collected for the three test componds

indicates a trend in that higher concentrations (50 150 M) of all three test componds

demonstrated mltiple peaks. This as confirmed ith the ShapiroFrancia test (Table 3.1),

here most of the 50 150 M data sets proed to be significantl nonnormal. Themltiple poplations that are obsered here ma hae been the reslt of differences

beteen the preparation of batches of NR de, bffers or test componds or differences

beteen cell passages, incbation times, instrment conditions etc. These reslts shold

not be considered definitie as the SEMs are also indicatie of reprodcibilit. Also, mean

ales obtained from the pooled indiidal assa reslts ielded good doseresponse cres

ith ales 0.86, hich is acceptable considering it is a nonlinear fit. This means that

the maimm amont of ariabilit that cold not be attribted for as 14% in the case of


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DDT, hereas it as 5% and 9% for DDE and DDD, respectiel. Taken collectiel, the

aboementioned indicates that the NRU assa ielded reprodcible reslts bt has its

limitations.

With regards to the effects of the test componds on cell iabilit, high concentrations of

the test componds ere necessar to significantl decrease cell iabilit after 24 h of

eposre. Significant ctotoicit as onl eident at concentrations aboe 10 M ith IC50

ales of 40 60 M. MedinaDia & Eliondo (2005), ho stdied the effects of DDT on

HepG2 iabilit, also reported the onset of toicit at concentrations higher than 10 M.

Reslts sggest that DDT (bt not DDE or DDD) ma indce celllar proliferation at sbtoic

concentrations. Althogh not significant, eposre to 5 M DDT reslted in a 5% increase in

cell nmber hen compared to the control. A possible eplanation for this can be fond in

the ork of Kiosaa . (2008), ho demonstrated the preglation of the cell

proliferationrelated genes 1, 2, 1, 1, and 2 in the liers of rats

eposed to DDT. These athors conclded that preglation of these genes ere facilitated

b actiation of the constittie androgen receptor b DDT, hich also reslted in the p

reglation of CYP3A4. As the eposre period as onl 24 h, it is possible that a longer

incbation time cold increase proliferation significantl. Celllar proliferation indced b

DDT has also been reported in MCF7 cells (breast adenocarcinoma) (Diel . 2002).

Srprisingl, this proliferatie effect decreased at a concentration of 10 M DDT, hich, in

the present std, as fond to be the concentration that did not adersel affect cell

iabilit. Diel .(2002) sggested that the decrease in proliferation might be de to the

onset of toicit at these concentrations. Rat mammar gland proliferation indced b DDT

has also been reported (Upalla ., 2005).

In contradiction to the reslts from the present std, Delesclse .(1998) obsered no

toicit in HepG2 cells eposed to DDT at concentrations as high as 100 M of DDT. Dehn

.(2005) reported an IC50of 1 mM for DDT in HepG2 cells, hich is approimatel 20 times

greater than the ale determined in the present std (54 M). This difference cold

possibl be attribted to the tenfold difference in cell densit sed beteen the to


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stdies: 2105 cells/ell (Dehn ., 2005) . 210

4 cells/ell, as ell as the ehicle

solent concentration. In the present std the ehicle solent, DMSO, did not eceed a

final concentration of 0.5% (/) compared to a 2.5% (/) concentration in the std b

Dehn . (2005). It has been reported that the addition of DMSO to cltre medim

improes the cell iabilit of isolated primar hepatoctes (Banic ., 2011), hich ma

also contribte to the difference in IC50ales beteen the to stdies.

The IC50 ales reported for DDT in other hepatocte cell cltres sch as primar

hepatoctes and HaCaT cells (rat lier) ere 250 M and 70 M, respectiel (Delesclse

. 1998). These IC50 ales and that reported in the present std sggest that different

hepatocte cltres ma not necessaril respond in the same a to DDT (and other test

componds for that matter). Primar cell cltres are considered the 'gold standard' bt as

alread mentioned, there are man drabacks in terms of feasibilit and predictabilit hen

tilising these hepatoctes.

At the highest concentration tested (150 M) of all three test componds some cells

remained iable. This cold demonstrate the resilience of hepatoctes bt ma also be a

draback of the assa that as emploed; in that complete cell death as not achieed.

Some of the NR de did bind to the ell srface, mimicking cell nmbers. Hoeer, becase

the releant blanks ere inclded in the assa setp, this shold hae compensated for an

nonspecific staining. Nonetheless, the assa ielded good doseresponse cres (Figre 3.4)

and the reslts ere reprodcible (aerage coefficient of ariation < 10), demonstrating the

reliabilit of the NRU assa.

Noticeable from Figre 3.4 is the difference in slope of the doseresponse cres of the test

componds alone compared to those of the cells that ere pretreated ith NAC. When

considering the IC50ales, NAC pretreatment did not protect against toicit indced b

an of the three test componds. Rather, it seemed to eacerbate it. On eamination of the

doseresponse cres this eacerbating effect can be seen at concentrations of 5 50 M of


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all three test componds. Hoeer, at higher concentrations (100 150 M), NAC pre

treatment appeared to hae alleiated some of the toicit indced b the three test

componds. Eactl ho this ma hae happened is not obios bt these reslts sggest

that different aspects of celllar phsiolog are inoled. On the one hand, loer

concentrations (5 10 M) of the test componds ma appear nontoic de to some

celllar response that allos the cells to cope ith the enobiotics. This crcial response

ma be conteracted b NAC, hich ill then decrease the resistance of the cells to the

eogenos stressor, casing the loss of cell iabilit obsered at these concentrations.

On the other hand, aboe some threshold concentration the cells ma not be able to

conteract the effect of the test componds, reslting in cell death. NAC ma be able to

conteract this mechanism of toicit, hich old alleiate some of the toicit seen

aboe this threshold concentration, as obsered for concentrations of 100 150 M of all

three test componds. No literatre as fond to compare/spport an of the NAC pre

treatment reslts obsered in the present std.

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