Comparison of the waterborne and dietary routes of exposure on the effects of Benzo(a)pyrene on

biotransformation pathways in Nile Tilapia (Oreochromis niloticus)

Rationale

• Polycyclic aromatic hydrocarbons (PAHs)– prevalent environmental pollutant/contaminant– aquatic sediments & waters associated with urbanized estuarine– volcanoes, forest fires, oil seeps– vehicle exhaust, power generation, oil pollution

*degree of toxicity influenced by: routes (ex. dietary and waterborne)doseduration of exposure

• Benzo(a)pyrene (BaP)– most studied PAHs– toxicity in aquatic organisms

Rationale

• Fish– bioindicators in aquatic environmental pollution assessment– exposure to PAH PAH metabolism in liver secretion of

metabolites into the bile stored in gall bladder excreted to intestinal tract

– Degree of exposure: PAH metabolites in bile , GST and EROD activities

• Nile Tilapia– economically important cultured species– well-established model in many toxicological studies

Statement of the Problem

(1) To evaluate differences in detoxification mechanism in Nile Tilapia after dietary and waterborne exposure to BaP;

(2) To assess biochemical effects of BaP by means of EROD and GST activities in liver, gill, intestine and BaP metabolites in bile.

*Additionally, fixed wavelength fluorescence (FF) was used to quantify BaP type metabolites as fluorescent aromatic compounds (FACs) in bile.

Methodology

Preparation of BaP stock solutions

(0.5, 1.25, 2.5, 5.0 g/L)administered directly to the

aquaria.

Waterborne ExposureXenobiotic exposure: nominal water concentrations (10, 25, 50 or 100 µg of BaP/L) for 14

days.

Preparation of BaP stock solutions

( 0.1 and 40 g/L) for 1st and 2nd assay resp.

Dietary Exposure

Xenobiotic exposure: 1st dietary assay – exposure to 1 and 10 µg

of BaP/g of food for 14 days.

Sampling

SamplingXenobiotic exposure: 2nd

dietary assay – exposure to 100 and 200 µg of BaP/g of

food for 21 days.

Food pellets were immersed in in BaP

stock solutions diluted in acetone.

Evaporation of acetone under air current for 24hr. Dried pellets

were stored at -20oC.

Methodology Waterborne and Dietary: Sampling

Anesthetization of fish on ice cold water. Then

sacrificed by decapitation.

Excision of liver, gills and intestine and collection of bile from gall bladder

using 1mL syringe.

Liver, gills, intestine and bile were stored at -80oC

after it was frozen in liquid nitrogen.

Biochemical Analysis: EROD activityHomogenization of

the tissues in ice cold buffer (Tris-HCl, KCl, pH 7.4).

Suspension of pellet produced in

buffer and centrifuged again.

Production of microsomal fraction of liver, gills and 1st

1/3 of intestine thru centrifugation.

Incubation of microsomal

suspension with ethoxyresorufin.

The enzymatic reaction was initiated by NADPH. Then

EROD activity measured by fluorometry at .

Determined by comparison to

resorufin standard curve.

Methodology

Biochemical Analysis: GST activity

Biochemical Analysis: Bap Metabolites in fish bile

Cytosolic fraction of the tissues were

obtained.

0.2 mL Rxn mixture + 0.1

mL sample

Production of reaction mixture: GSH + phosphate

buffer + CNDB

Measurement of GST activity at 340

nm. (at every 20s during 1st 5 min)

Dilution of bile samples from both routes –

1:1000 in EtOH.

Further dilution of water exposed

(1:104 and 1:105).

FF values were

expressed as a.f.u.

Measurement of GST activity at 340

nm. (at every 20s during 1st 5 min)

Results and Discussion Waterborne Exposure: EROD activities

Fig 1. EROD activities in liver (a), gill (b), and intestine(c) after waterborne exposure to BaP.

EROD activity in liver, gills and intestine increased during exposure period.

Waterborne exposure Bap metabolism in liver(CYP1A enzyme) and also in extrahepatic tissue.

Strong gill EROD introduction indicates rapid absorption

gills work in the first-pass metabolism of BaP

Extrahepatic tissues more sensitive than liver also useful to measure EROD activity being involved in first-pass metabolism

Results and Discussion Waterborne Exposure: GST activities

Fig 2. GST activities in liver (a), gill (b), and intestine(c) after waterborne exposure to BaP.

Most significant result induction of EROD act. in intestine after exposure to highest conc.

Dietary BaP moderate CYP1A staining in liver High in gut mucosal epithelium

(intestinal tract) Due to partial BaP metabolism in intestine limiting

parent compound to reach liver for hepatic metabolism

Decrease hepatic EROD activity not physiologically relevant

Results and Discussion Waterborne and Dietary Exposure: BaP type Metabolites

Fig 3. BaP type metabolites in liver (a), gill (b), and intestine(c) after waterborne exposure to BaP.

BaP Metabolites in bile increased Highly correlated with all tissues

Liver correlation leads to pathway of high hepatic metabolism by CYP1A enzymes

Dietary BaP Metabolites formed in intestine reabsorbed into blood and excreted into bile

BaP metabolites more elevated after water exposure

Indicates that levels of biliary metabolites gives and indication of the main route of exposure

Results and Discussion Dietary Exposure: 1st and 2nd exposure EROD activity

Fig4. (left) first dietary exposure Fig 6.(right) second dietary

exposure

In liver EROD activity reduction

Gill EROD activity didn’t change

Significant increase in intestine EROD act.

Therefore, dietary EROD activity is seen only in intestine

Results and Discussion Dietary Exposure: 1st and 2nd exposure GST

activity

Fig5. (left) first dietary exposure Fig 7.(right) second dietary

exposure

Phase II enzymes GST did not change in liver nor in gill but only in intestine

Phase II enzymes GST with CDNB as substrate low sensibility to presence of BaP

It shouldn’t be applied as a biomarker of exposure to this pollutant

Conclusion

• This study has shown that:

(1) The disposition and effects of BaP in biotransformation pathways in Nile Tilapia depend on the route of exposure to the contaminant.

(2) Waterborne exposure resulted in an induction of EROD in liver, gill and intestine while in Dietary exposure route induction was only seen in intestine.

• EROD activity is a reliable biomarker.• Besides liver, gills and intestine should also be considered in

biomonitoring studies.• BaP metabolites are good reflectors of exposure despite route.• Levels of metabolites - indicative of route since water exposure lead to

much higher metabolites than dietary. • GST activity with CDNB not reliable biomarker regardless of route.