CHAPTER 8

r y

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Study on the ameliorative potential of gentisic acid on

chemically and UVB mediated biochemical

alterations in Swiss mice

Gemtisic add

Primary prevention includes the cessation o f contact with the causative factors o f a disease.

Practically, the feasibility o f such a.n approach is poor. On the other hand, secondaiy prevention

or what is more coiiimoniy called as cheraoprevention is quite successftil and has shown

promising results against carcinogenesis. The chemopreventive agents might belong to entirely

unrelated class o f chemicals Ice coumarins, azo dyes, sul&i- compounds, flavones, phenols,

indoles, retinoids, tochopherols, seleniiim compounds and protease inhibitors (Prochaska et al

1985).

In one of the eai'lier studies (described in Ciiapter 5), the chemopreventive effect of Hibiscus

rosa simnsis extract on the niarker parameters of tiimor promotion and, oxidative stress is

shown. Oxidative stress, ODC activity and DNA synthesis are invariably induced in case o f

tumor promotion as is also obseiTed in case of single topical applications of different promoters

such as 12™0"tetra decanoyl phorboi-13-acetate, benzoyl peroxide and ultraviolet radiations.

These short-term efiects o f TP A, BPO and UYR are o f great significance as they represent a

part of the complex process that talces place over a course o f time. If these short-term alterations

can be blocked then cancer formation can be delayed to a certain extent. Out o f the three stages

of caiicer, initiation is an irreversible stage while progi'ession mai'ks the last stage of cancer

events, so the only step, which can te promising in the prevention o f cancer, is tumor promotion

that is reversible in early stages (DiGiovanni 1992). Tumor promotion stage features only

epigenetic changes tliat include alterations in the biochemistry and histopathology o f the tissue.

The biochemical parameters associated with tumor promotion include alterations in, above said

parameters such as the activities o f certain enzymes; enhanced sjmthesis o f DNA, KNA and

proteins; oxidative insult to the cells (Perchellet and Perchellet 1989).

135

Geniisic acid

With the application o f the toxicant alone, the level of cellular antioxidant, glutathione is also

depleted along with the depletion of its metabolizing enzymes. The increased level of MDA is

an indicator o f increased peiwddation o f Hpids. Therefore, MDA and eniianced hydrogen

peroxide formation in the TP A, BPO and UVR treated groups act as direct indicators of

oxidative damage that might lead to genotoxicity or cytotoxicity in cells (Sun 1990). The extract

of Hibiscus rosa sinensis used in one of the studies described earlier showed promising results

against two-stage skin carcinogenesis. Keeping this in view, we analyzed the effects of one of

its chemical constituents. The extract of the plant possesses various compounds including

quercetin, cai'otene, niacin, ribollavin, malvalic acid, gentisic acid, margaric acid and iauric acid

(Ross 1999). Since quercetin, carotene, niacin have been studied extensively our aim was to find

out whether or not other chemical constituents also contribute to the modulatory activities of

medicinal plants. Mostly, the research work focuses on the effects o f the major principle or the

active constituent o f the plant extract and viitually veiy few minor constituents di'aw the

attention o f the researchers. However, we were particularly interested in one o f the minor

constituent, gentisic acid of the plant studied earlier. Chemically it is 2, 5»dihydroxy benzoic

acid (2, 5 DHBA), wliich is a phenolic acid. It is basically an adduct product of salicylic acid

and hydroxyl radical, therefore, is generated in vivo as well. Besides, Radtke et al (1998) have

shown that the total sum of hydroxybenzoic acids amounts to 11 mg/ day with 75% of salicylic

acid intake by plant derived foodstuff including fruits and juices. While, Hinz et al report that it

accounts for less than 5% of the ingested salicylic acid (Hinz et al 2000). Chronic arthritis

especially in children leads to nutritional impairments, which may start with food allergies and

extend to chronic malnutrition. In such cases the biotransformation of salicylic acid to gentisic

acid is adversely alfected and the levels o f gentisic acid is depleted (Lares-Asseff et al 1999),

136

Gentisk acid

Gentisic add is also |3resent fcis normal constituent of serum aiid therelbre it is important to find

answers for questions regai'diiig its role in the promotion of health (Patereon et al 1998). Most

phenolic acids have aiitioxidajit and aiiticarcinogetiic properties. It is noteworthy here that

although the salicylate metabolized to gentisic acid is very less, it has been shov^n to be

iiivolved in the antioxidaiit activities and it is also formed during inflammatory conditions by the

polynmorphoniiclear ieiicocytes that produce reactive oxygen radicals (Hinz et al 2000). Other

scientists have also shown evidences that it possesses antioxidant, anti-inflafiimatoiy and anti-

mutagenic properties (Ivi'izkova et al 2000, Uhrig et al 2000).

Based on these facts v^e speculated the role o f gentisic acid against TPA and the combination of

BPO and UVB induced tumor promotion response and oxidative stress in mice and hence

proceeded with the present study. The assessment o f antioxidant activity and anti-promotional

mai'kers v^ere assessed using the saine pai-ameters as described in Chapters 4-7.

TREATMENT PROTOCOL

Sfiort-teras studies wttti geEitisIc acid against BPO treatment & UVB Irradtatw i lidueecl

fjlocliteinkal ebaisges

Groffips Tre^taiest RegimeH

Day 1 Day 2 Day 3

! Vehicle Veliide VehicleII ■ Vehicle Vehicle BPO + UVBin Vehicle VeMcle BPOIV Yehlde Vehicle OTBV Gentisic add (Dl) Gentisic acid (Dl) Geatfeic acid (Dl) + BPO + 0VBVI Gentisk acid (D2) GeHtlsk add (D2) Gemtislc acid (D2) + BPO + UVBVII Gemtlsic add (D2) Geitlsk acid (D2) Gentisic aeid (D2)

137

'j;sasssss3mzs:fysmmz3fsmxm!sss^ Gentisic acid

Vehicle [Acetone: Methanol, 9:1] == 0.2ml/aMmal, Gentisic acid (D i) = 2.0}ig /0.2ml vehicle/

animal, Gentisic acid (D2) ^ 4.0|iig /0.2ml vehicle/ animal Doses o f BPO and UVB; age and

number o f the animals; the time period between the gentisic acid pretreatment and BPO +

UVB exposure; and the time o f sacrifice o f all the animals for the estimation o f different

pai’ameters was similar to that described in Chapter 4.

Sfiort-term stiicMes witli gesstlsic acid against TPA indiiced blodicmkal cisiiges

Groups Treistiifieut Regimen

Bay 1 Day 2 Day 3

1 Vehicle Veliicle VehicleII Vehicle Velilde TFAHI Geetfelc acid (Dl) Gentisic acid (Dl) Geiifeic acid (Bl) +TPAIV Geatfeic acid (1)2) Gesitisic acid (D2) Gentisic acid (D2) + TPAV Gentisic sicW (D2) Geiitfetc acid (I>2) Gentisic acid (D2)

Vehicle, Gentisic acid (D l) and Gentisic acid (D2) are same as described above. TPA =

20nmol/0.2ml/animaL Each group contains six mice. TPA = 20nmo]/0.2ml/animal. Sequence o f

treatments on the third day aiid the time of sacrifice o f the animals were same as that described

in Chapter 4.

RESULTS

Effect o f gemtmc a d d m BFO & UVB mediated oxidative stress and kyperprolifer&tmm

response

Figure 1 depicts effect o f prophylactic treatment o f gentisic acid on BPO ti’eated and UVB

mediated depletion in reduced glutathione and glutathione reductase in murine skin. BPO and

UVB caused 65 and 71% in reduced glutathione and glutathione reductase respectively.

138

— ______ ______ _____ ^ittisic (tad

Prophylaxis with geiitisic add caused 3-11% and 15-20% recovery at tow and high doses

respectively in case o f reduced glutathione and glutatliiooe reductase.

Figui’e 2 shows effect o f prophylaxis of gentisic acid on BPO treated and IJVB mediated

depletion in cutaneous gliitathione-S“tra,Bsferase and quinone reductase in mice. BPO plus IJVB

irradiation caused 65 and 70% reduction in glutathione-S-transferase and quinone reductase

respectively. Prophylaxis with gentisic acid caused 7-21 and 16-28% at both the doses.

Figure 3 shows eliect of pretreatment with gentisic acid on BPO and DVB mediated depletion

of catalase, glutatMone peroxidase and gIucose-6-phosphate dehydrogenase in skin. The

depletion observed in case o f catalase, glutathione peroxidase and glucose-6-phosphate

dehydrogenase was 58, 69 and 68%. Pretreatment with gentisic acid, however, recovered their

activities by 4-10 and 14-21% at low and high doses respectively.

Figure 4 shows that treatment with BPO and IJVB induced hydrogen peroxide fonnation

(121%) and lipid peroxidation (231%) in murine skin whereas pretreatment with gentisic acid

caused 10-14%o and 58-71% reduction respectively in the levels o f the above said parameters.

Figure 5 depicts the effect o f gentisic acid in the attenuation o f BPO and UVB induced ODC

activity and DNA synthesis. The activity of ODC and the synthesis o f DNA increased to 261

and 204% in BPO treated and UVB irradiated group; 231 and 184% in the only BPO treated

group; and 150 and 126% in only UVB treated group respectively. While, pretreatment of

gentisic acid caused 131 and 75% decline in the elevated ODC activity and 40-68% inhibition in

case of DNA synthesis respectively.

Effect o f gentisic ad d m TPA mediated oxidaiive stress and hyperproiifemtion response

Figure 6 shows the moulatoiy effect o f gentisic acid on TPA mediated depletion of cutaneous

reduced glutathione content, glutathione-S-transferase and glutathione reductase. Only TPA

139

______ __ _ __ G&tttiS i£ (iCiSSr

application to mice caused 60, 47 and 47% decrease in reduced glutathione content, giutatiiione“

S“transferase and glutathione reductase respectively. Pretreatment with gentisic acid however,

caused 10-21% recovery at low dose and 18“28% recoveiy at higli dose.

Figure 7 exliibits pretreatment efiect of gentisic acid on TPA mediated depletion of cataiase,

glutathione peroxidase and glucose-6-phosphate dehydrogenase in skin. The activities of

catalase, GPx and G6PD were significantly depleted on application of TPA to the dorsal shaven

portions of the mice sldns to 31, 58 and 48% respectively, Pretreatmeot with gentisic acid on the

other hand, caused 8«15% recovery at low dose and 22-40% recovery at iiigh dose.

Figure 8 shows that malondiaidehyde formation (207%) and H2O2 content (177%) were

significantly elevated in. TPA treated group. Gentisic acid pretreatment caused 44 & 56%

decrease in MDA formation and caused 27 & 39% recovery in H2 O2 content in a dose

dependent iiianner-

Eflfect of gentisic acid on the induction of ornithine decarboxylase and DNA synthesis in mouse

sldn by TPA is shown in Figure 9. There was 5.8-foid increase in ODC activity and 2.4-fold

increase in thymidine incorporation in DNA on treatment of animals with single topical

application of TPA on mice skins. The activity of ODC was restored in case of gentisic acid but

not in a dose dependent manner. On pretreatment with gentisic acid the recovery in

thymidine incorporation into DNA was 77 & 88%, which was dose dependent.

DISCUSSION

Gentisic acid is one o f the components o f Hibiscus rosa sinensis extract, plant that has already

been described in Chapter 5, Other aspect that makes it more interesting for study is the fact that

it can tel generated in vivo also. In most o f the studies, the formation of hydroxyl radical is

quantitated in terms o f the increased amounts o f 2,3 and 2,5 hydroxybenzoic acid that are

140

___________

hydroxylative products of salicylic acid (Prasad 1997). It is a second Une metabolite of aspiiin

(Exner et al 2000). It is also used in cosmetics as a skin-whitening agent (Bian et al 2003).

Gentisic acid (2,5 hydroxybenzoic acid), on its part, has been shown to possess antioxidant

properties in various studies as it scavenges free radicals (Liu and Edwards 2001). It

significantiy inhibits the formation of PGE2 at concentrations in which it is formed in aifhritis

(free-radical mediated disease) patients (Hinz et al 2000). ft lias anti-mntagenic activity (Hinz et

al 2000) as well, allhough no substantial amount o f work has been done to check the eiScacy o f

this compound against tumor promotion. In the current study also, we have evaluated the

potential of the test agent using two mechanisms of anti-tumor promotion i.e. inhibition of

oxidative stress aod polyamine biosynthetic parameters.

The results o f the present study show that gentisic acid potentially prevents the oxidative

damage induced by different promoters. Ail the thi’ee agents used in this study induce

oxidative stress. During such a stress, superoxide anion is the &st ROS to be generated and if

it is not prevented Iroiii generation it leads to the formation o f highly reactive species,

hydroxyl radicals, that attack proteins aod lipid membranes causing cell damage or genetic

alterations. Lipid peroxidation and hydrogen peroxide are therefore, the markers for

assessing the extent o f damage in cells (Zhao et al 2000). Gentisic acid helps in the

restoration o f intracellular antioxidant, reduced glutathione and antioxidant enzymes together

with the inhibition of lipid peroxidation and hydrogen peroxide generation in a dose

dependent manner. Gentisic acid like other modulators successfully prevented oxidative

stress induced not only by the combination o f BPO and UVB but also by the individual

strong tumor promoter, TP A. It restored the cellular glutathione content and the activities o f

antioxidant enzymes like catalase and glutathione peroxidase. Additionally, it also inhibited

141

entisic acid

the epidermal peroxidation o f lipids and the formation of hydrogen peroxide that would

otherwise have lead to the formation o f reactive species. Several naturally occurring

chemopreventive agents also infiibit the formation o f H2 O2 and oxidized DNA bases (Huang

et a! 1997). A lot o f chemopreventive agents act by inhibiting the formation o f hydrogen

peroxide (Wei and Frenkel 1993, Wei et al 1,993). Exner et al (2000) have reported that both

salicylate and gentisic acid, have the ability to inhibit LDL oxidation induced by superoxide

or by nitric oxide. Wliile some o f the phenolic acids like 23-DHBA form complex with the

metal ions, 2,5-DHBA scavenges the fi'ee radicals. Thus, gentisic acid suppresses promoter-

mediated oxidative insult iii mouse epidermis. It is believed that for chemoprevention of

cancer, ioMbition of tumor promotion is a better strategy as compared to tumor initiation.

This is because tum.or initiation is short and iiTeversible event whereas tumor promotion is a

long process wherein many biochemical and histological alterations occur that are reversible

during early phases (Zhao et at 1999). It is a well-known fact that oxidants ai-e believed to

have a crucial role in all the stages o f cancer. Many modulators in other studies and even in

our own previous lab studies (Saleem et al 2001, Sultana et al 2003) have shown that agents,

which have the ability to prevent oxidative stress are also capable of inhibiting tumor

promotion related biochemical alterations e.g. ODC activity. With this view in mind, the

compound, gentisic acid was forther tested for its anti-tumor promoting ability against TPA

and the combination o f BPO with UVB. Second mechanism of action that was analyzed for

the study o f gentisic acid was the inhibition o f ODC activity. Induction of ODC activity is

considered to be an important biochemical marker o f tumor promotion as it is associated with

cell proliferation. The data o f the current study does not seem to support the role o f inhibition

of polyamine biosynthetic pathway by the compound. Though gentisic acid appears to have

142

Gentlsk acid

modulated or suppressed the ODC activity, the suppression was not dose dependent. It

suggests that gentisic acid does not have a specific effect oo the modulation o f ODC activity

induced either by TPA or by the combination o f BPO and UVB especially not by this mode

of action. Surprisingly though, it caused a dose dependent inhibition of unscheduled, DNA

synthesis. Enkvetchakul et al have also reported some paradoxical facts about another

compound,, sphingosine, which exhibits inhibitory effect on ODC activity induced by TPA in

one study while in the other study, it enliances TPA-promoted tumorigenesis (Enkvetchakul

et al 1989, Enlcvetchakul et a! 1992). There are certain other agents also that do not work by

this mechafflsm o f anti-'tumor promotion action but are potent anti-tumor agents (Wei et al

1998) although a positive role played by any agent in the amelioration o f this marker

parameter invariably acts as a potential anti-tumor agent. Various monocyclic phenolic

compounds (especially BHA and BHT) have been reviewed for their anticarcinogenic

activity (Williams et al 2002) and the compound, gentisic acid also belongs to the category of

monophenolic compounds.

The present study also gives an insight into the mechanism of action o f Hibiscus rasa sinensis

extract against cancer since lx)th the H. sinensis and its chemical constituent, gentisic acid were

able to inhibit TPA, BPO and UVB induced unscheduled DNA synthesis. The unscheduled

DNA synthesis was dose dependentiy inhibited by gentisic acid. On the other hand, when we

compare the recovery in unscheduled DNA synthesis in case o f H. sinensis extract (Chapter 5),

we observe that H. sinensis showed more pronounced prevention as compared to that caused by

gentisic acid. Tliis suggests that besides gentisic acid other components too have a crucial role in

anti-tumor promoting activity of H. sinensis extract. This tact is fijrther strengthened by

observations that H. sinensis extract causes dose dependent inhibition of the ODC, the rate-

143

_________ Gentisic acid

Ijfiiiting e n : ^ e in timior proiiiotioa However, with gentisic acid there surely was statistically

significant inhibition o f ODC activity but the results obtained were not dose dependent,

therefore, we cannot say that gentisic acid has a key role in protection against tumor promotion

response via this njechanism. Since oxidative stress is impiicated in the etioiogy o f many

diseases including initiation, promotion and progression, we can say that gentisic acid might

have the ability to interfere with the process of carcinogenesis as an antioxidant. However,

owing to its anti-oxidant properties it must have some indirect role in the disease prevention

process. Since gentisic acid has sufficient anti-oxidant activity and is also shown to possess

inhibitory activities against mutagenesis and cyclooxygenase activity it nnight play some role in

the suppression o f inflamiTiation and might have a role on mutations as cooperation o f genetic

defects and sustained high levels of (3DC and polyamine levels are thought to be required for

neoplastic transformation and clonal expansion of tumor cells. Different kinds of herbs

{Actinidia chinensis, Artemisia lavendulaefoUa, Crotalaria sessijhra, Pmnella vulgaris, Paris

polyphylla and Ampelopsis hrevipedmculata) that are used as crude medicine for cancer have

been shown to possess antimutagenic potential (Lee and Lin 1988).

The fact that gentisic acid protects against oxidative stress, dimimshes incorporation of [^H]

thymidine into DNA, proves that it has some modulatory activity but since it was unable to

dose-dependently inhibit ODC activity., we cannot say that it has key role in protection against

tumor promotion. Also, to the best o f our knowledge this compound has not been tested

against cancer formation. We can nevertheless, conclude that fiirther studies employing

different aspects or mechanisms of chemopreventive action should be done to find out whether

it has any other mode o f action for the prevention of tumor promotion or it has no role in tliis

144

W 8 Gentisic acid

preventable stage o f tumor formation. Tims, there is a long way to go before any conclusion can

be drawn regai’ding the chemopreventive potential of gentisic acid.

145

J _______ _________ _____Gentisie acid

F O O T N O T E S A M D F IG U R E L E G E N B S

Figure 1Role o f gentisie acid in BPO and UVB mediated depletion o f cutaneous (a) reduced glutathione content and (b) gliitatliiooe reductase

Each value represents mean ± SE o f six animals. M ii p< 0.001 as compared to vehicle treated control group. p< 0.001 and p< 0.01 when compared with BPO treated and UVB irradiated group. D-1 and D-2 represent topical applications o f 2,0 and 4.0 |ug gentisie acid/ 0.2ml vehicle / animal.

Modulation o f BPO and U VB depleted (a) glutathione- S~ transferase and (b) quinone reductase by gentisie acid in mouse skin

Each value represents mean ± SE o f six animals. ### p< 0.001, ## p< O.Ol and H p< 0.05 as compared to vehicle treated control group, p< 0.01 and p< 0.05 when compared with BPO treated and UVB irradiated group. D-1 and D-2 represent topical applications of 2.0 and 4.0 pg gentisie acid/ 0.2ml vehicle / animal.

Effect of preti’eatment with gentisie acid on BPO and UVB mediated depletion o f (a) catalase, (b) glutathione peroxidase and (c) glucose-6-phosphate dehydrogenase in skin

Each value represents mean ± SE o f six animals. ### p< 0.001, ## p< 0.01 and # p< 0.05 as compared to vehicle treated control group, p< 0.01 and p< 0.05 when compai'ed with BPO treated and UVB irradiated group. D~i and D-2 represent topical applications of 2,0 and 4.0 fig gentisie acid/ 0.2ml vehicle / animal.

Effect of pretreatment with gentisie acid on BPO and UVB induced (a) hydrogen peroxide formation and (b) iipid peroxidation in murine skin

Each value represents mean ± SE o f six animals. ### p< 0.001 and # p< 0.05 as compared to vehicle treated control group, p< 0.01 and * p< 0.05 when compared with BPO treated and UVB irradiated group. D~1 and D-2 represent topical applications o f 2.0 and 4.0 lag gentisie acid/ 0.2ml vehicle / animal.

Study of the effect o f gentisie acid in the attenuation o f BPO and UVB induced cutaneous ODC activity and DNA synthesis

Each value represents mean ± SE o f six animals. ### p< 0.001 and # p< 0.05 as compared to vehicle treated control group. p< 0.001, p< 0.01 and p< 0.05 when

Geniisic acid

coinpar€!d with BPO treated and UVB irradiated group. D -l and D-2 represent topical applications o f 2.0 and 4.0 ig gentisic acid/ 0.2ml veliicle / animal

Modulatory effect o f gentisic acid on TPA mediated depietioii o f cutaneous (a) reduced glutathione content (b) glutathione-S-transferase and (c) glutathione reductase

Each value represents mean ± SE o f six animals, p< 0.001 and ## p< 0 .01 as compared to vehicle treated control group. p< 0.001, p< 0.01 and p< 0.05 when compared with TPA treated group. D-1 and D-2 represent topical applications of 2.0 and4.0 Jig gentisic acid/ 0.2ml vehicle / animal

Pretreatment efliect o f gentisic acid on TPA mediated depletion o f (a) catalase, (b) glutathione peroxidase and (c) gIucose-6-phosphate dehydrogenase in skin

Each value represents mean ± SE o f six animals. ### p< 0.001 and # p< 0.05 as compared to vehicle treated control group. p< 0.001, p< 0,01 and p< 0,05 when compared witli TPA treated group. D-1 and D-2 represent topical applications o f 2,0 and4.0 .tg gentisic acid/ 0.2ml vehicle / animal

Effect o f gentisic acid pretreatment on TPA induced (a) hydrogen peroxide formation and (b) lipid peroxidation in mouse skin

Each value represents mean ± SE o f six animals. ### p< 0.001 as compared to vehicle treated control group. p< 0.01 and * p< 0.05 when compared with TPA treated group.D 4 and D~2 represent topical applications o f 2.0 and 4.0 fig gentisic acid/ 0.2ml vehicle / animal

Figure 9Pretreatment of gentisic acid on TPA induced cutaneous ODC activity and radiolabelled thymidine incorporation

Each value represents mean ± SE o f six animals. ### p< 0.001 when compared with vehicle treated control group. *** p< 0.001 and p< 0.01 when compared with TPA treated group. D1 and D2 represent topical applications o f 2.0 and 4.0 fig gentisic acid/ 0,2nil veliicle / animal

Chapter 8 Gentisic acid

(a)

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Chapter 8 Gentisic acid

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Chapter 8 Gentisic acid

(a)

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i•cIO3f"SEe

Figure 8Effect of gentisic acid pretreatment on TPA

induced (a) hydrogen peroxide formation and (b) lipid peroxidation in mouse skin

500TTtttt

I450 400 350 I 300 250 200

150 100

50 0

2 3 4

T reatment groups

IV ^ C* 3II I I 1e

6 1

5

oEc 0

n FT n

NS

2 3Treatment groups

□ Only Vehicle□ Gcntisic acid (Dl) + TPA■ Only Gentisic acid (D2)

□ Onlv TPA■ Gentisic acid (D2) + TPA

Chapter 8 Gentisic acid

Figure 9Pretreatment of gentisic acid on TPA induced cutaneous ODC activity and DNA synthesis

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- § 5000

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2000

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0 I

- L

2 3 4

Treatment groups

C9L.o^ 5© z a G

!i

700

600

500

400

300

200

100

0

2 3 4

Treatment groups

□ Only Vehicle□ Gentisic acid (Dl) + TPA□ Gentisic acid (D2) ____

□ Only TPA■ Gentisic acid (D2) + TPA