PETROLEUM INCREASES SENSITIVITY TO CHLORPYRIFOS IN RAINBOW TROUT LIVER JULIETA S. DE ANNA1, LEONARDO R. LEGGIERI1, WALTER D. TORRES2 ANDRÉS VENTURINO3, CARLOS M. LUQUET1* 1 LAB.
ECOTOXICOLOGÍA ACUÁTICA (INIBIOMA-UNCO-CONICET), 2 CENTRO DE ECOLOGÍA APLICADA DEL NEUQUÉN, 3 UNIVERSIDAD NACIONAL DEL COMAHUE. NEUQUÉN
*
[email protected]
INTRODUCTION Fish could be simultaneously exposed to various pollutants, resulting in overlapped effects on their detoxification systems. It is known that CYP1A expression level is induced in fish exposed to polycyclic aromatic hydrocarbons from petroleum and that chlorpyrifos (CPF) is activated to chlorpyrifos-oxon by CYP1A activity [1]. The CPF-oxon inhibits carboxylesterases (CEs) and acetylcholinesterase (AChEs) by binding to non-substrate sites [2]. Thus, preexposure to petroleum before pesticide exposure of fish to would lead to enhanced CPF activation with the consequent increase in toxicity. We hypothesize that water accommodated fraction of petroleum (WAF) enhances sensitivity to CPF in rainbow trout.
FACTORIAL DESIGN
Oncorhynchus mykiss
METHODS
WAF preparation: 4.75g crude oil / L river water [3] Exposed juvenile trout to 5% WAF in vivo for 48 h (n = 6), 1 fish/10 L
Treatment
Extirpated livers exposed to 20 µg/L CPF ex vivo for 1 h
CEs: p-nitrophenyl butyrate as substrate, A400nm , 1‘. Enzyme activities:
2 Factor × 2 Level
WAF 1
WAF 0
CPF 0
WAF& CPF
WAF - SC
C - CPF
C - SC
GST: CDNB and GSH as substrates, A340nm , 5‘.
AChEs (nmol /min /mg prot)
AChEs activity was inhibited by CPF irrespectively of WAF pre-treatment (*P < 0.05) 24 16
* ▼45%
* ▼24%
8 0
C-SC
C-CPF
WAF-SC WAF-CPF
GSTs (nmol /min /mg prot)
GST activity was not affected by WAF and was increased by CPF in both pre-treatments (F = 8.14, **P < 0.01) 6
**
▲30%
** ▲57%
4
AChEs: 1.5 mmol /L acetylthioch o- line iodide as substrate, A 412 nm, 1’
The inhibition rate of CEs activity by CPF was higher in WAF pre- treated trout than in control trout (t = 3.057, *P < 0.05) CEs (nmol /min /mg prot)
Pre-treatment
CPF 1
Proteins by Bradford
Homogenized with Tris-HCl 20 mM, pH 7.5, EDTA 0.5 mM, 11000 xg, 15', 4°C
15
p > 0.05
*
10
▲15%
*
5 0
C-SC
CONCLUSIONS
C-CPF
WAF-SC WAF-CPF
WAF pre-treatment did not affect the response of liver AChEs and GST to CPF. WAF and CPF had additive inhibitory effects on CEs activity, since there is no significant
2 0
C-SC
C-CPF
WAF-SC WAF-CPF
ACKNOWLEDGMENT This work was supported by grant ANPCYT PICT 2013-1415 and CONICET PIP 11220130100529CO (Argentina) We thank the staff of the CEAN (Neuquén, Argentina) for supplying fish. J.S. De Anna has a doctoral fellowship from CONICET.
interaction
between
the
effects
pollutants.
However,
a
separate
of
both
analysis
suggests increased sensitivity to CPF, which should be further investigated. REFERENCES [1] Fukuto 1990. Environ. Health. Perspect. 87:245–254; [2] Fulton & Key 2001. Environ Toxicol Chem. 20:37-45; [3] Singer et al. 2000. Mar. Pollut. Bull. 40:1007-1016.
