Alterations in rabbit aorta induced by types I and II pyrethroids

Environmental Toxicology and Pharmacology 23 (2007) 250–253

Short communication

Alterations in rabbit aorta induced by types I and II pyrethroids Cinzia Nasuti a,∗ , Franco Cantalamessa a , Craig J. Daly b , John C. McGrath b a

Department of Experimental Medicine and Public Health, University of Camerino, Via M. Scalzino 3, 62032 Camerino (MC), Italy b Autonomic Physiology Unit, Division of Neuroscience & Biomedical Systems, Institute of Biomedical & Life Sciences, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK Received 11 May 2006; received in revised form 10 August 2006; accepted 14 August 2006 Available online 26 August 2006

Abstract Since pyrethroids are involved in reactive oxygen species production and no investigations have yet been performed on smooth muscle cell integrity, we studied the influence of permethrin- and cypermethrin-treatment on rabbit aorta using confocal laser scanning fluorescence microscopy, which allows cell viability to be assessed within the wall of living rabbit aorta. The data obtained show that the pyrethroid-treatment (10–100 ␮M) impairs the smooth muscle cell viability. A double-labeling protocol allowed us to distinguish cytotoxic effects of permethrin- and cypermethrintreatment in aortic rings. In conclusion, permethrin seems to induce more oxidative stress on the aorta wall than that cypermethrin does. © 2006 Elsevier B.V. All rights reserved. Keywords: Pyrethroids; Aorta; Cell viability; Smooth muscle cells; Rabbit; Laser scanning confocal microscopy

1. Introduction Pyrethroids are strong insecticides, which have low mammalian toxicity and do not persist for a long time in the environment. However, the liberal use of pyrethroids increases the risk of environmental contamination and intoxication of non-target organisms in different ecosystems (Bolognesi, 2003). Pyrethroid pesticides are divided into two groups according to their chemical structures: type I pyrethroids are devoid of a cyano moiety at the ␣-position (i.e. permethrin, PERM), while type II pyrethroids have an ␣-cyano moiety (i.e. cypermethrin, CY). With regard to the mechanisms of action responsible for toxicity, pyrethroids increase sodium entry, resulting in the delay of channel closure and membrane repolarization, which leads to blocking of nerve conduction (Vijverberg and Van den Bercken, 1990; Narahashi, 1992). Besides this neurotoxic effect, these insecticides are involved in the production of oxidative stress (Kale et al., 1999). The oxidative stress, apart from its direct cytotoxicity, has an additive or accelerating effect on the neurotoxicity of Na+ channel blockers.

∗

Corresponding author. Tel.: +39 0737 403318/403302; fax: +39 0737 630618. E-mail address: [email protected] (C. Nasuti). 1382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2006.08.008

Our previous study showed that CY and PERM, administered by gavage for 2 months in rats, induced alterations in erythrocytes, producing increased lipid peroxidation and consequently reduction of plasma membrane fluidity in the hydrophobic region of the bilayer where these pyrethroids are preferentially localized. A different effect of CY and PERM could be observed: PERM induced more oxidative stress than CY did (Gabbianelli et al., 2004). The aim of the present study was to evaluate the effect of CY (type II) and PERM (type I) on rabbit aorta by using in vitro tests and image analysis. Data are presented on the effect of pyrethroids on cell viability which is influenced, as reported in the literature, by increased production of free radicals (Miller et al., 1998). We have used laser scanning confocal microscopy (LSCM) to image aortic segments at the cellular level. The fluorescent nuclear stain, ethidium homodimer (EH), was used to identify damaged cells. 2. Materials and methods 2.1. Chemicals Analytical grade (75: 25, trans: cis; 94% purity) 3-phenoxybenzyl(1R,S)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxyl-ate (PERM) was generously donated by Dr. A. Stefanini of ACTIVA, Milan, Italy. (R,S)-␣-cyano-(3-phenoxy-phenyl)-methyl-3-(2,2-dichloro-vinyl)-2,2dimethyl-cyclo-propan-carboxylate (98% purity) (CY) was obtained from

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Sigma–Aldrich (Germany). Syto 13 green fluorescent nucleic acid stains (Ex. 488, Em. 509) and ethidium homodimer (Ex. 528, Em. 617) came from Molecular Probes (Paisley, UK). Other reagents used in this study were of analytical grade.

out using the one-way analysis of variance followed by Newman–Keuls test. A value of P < 0.05 was considered statistically significant.

2.2. Animals

3.1. Quantitative analysis of SMC permeabilized by PERM and CY

Experiments were carried out on male New Zealand White rabbits (3–4.5 kg). The animals were housed in plastic (Makrolon) cages in a temperature-controlled room (21 ± 5 ◦ C) and maintained on a laboratory diet and water ad libitum. The light/dark cycle was from 07:00 to 19:00 h. The procedures were undertaken in accordance with the Animals (Scientific Procedures) Act 1986 and the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996).

2.3. Tissues and solutions preparation The animals were sacrificed by an overdose of pentobarbitone (100 mg kg−1 ) into the ear vein. The thoracic aorta was then carefully removed and cleaned of fat and connective tissue under a dissecting microscope. Care was taken not to damage the intimal surface of the aorta. Tissues were used that day. The aorta was cut into transverse rings (2.5 mm wide), and those in segments approximately 2 mm in length. The segments were placed on a light-protected chamber (at 37 ◦ C) in which the Krebs’ solution can be bubbled with 95% O2 /5% CO during the staining period without damaging the aortic tissues. CY and PERM were dissolved in ethylic alcohol (in the medium <5%, v/v) and solutions from 100 to 1 ␮M were prepared.

2.4. Vessel imaging and laser scanning confocal microscopy The nuclear dye EH was used to show oxidative damage (membrane permeation) caused by PERM or CY. EH is a hydrophilic membrane-impermeant dye and will only label the nuclei of those cells which have a permeable membrane (Daly et al., 1992). If the pyrethroid-treatment selectively permeabilizes the smooth muscle cells, the damage is indicated by the uptake of EH. It intercalates with DNA and fluoresces red upon excitation thereby defining the nuclei of cells with permeabilised membranes. Syto 13 is a membrane-permeant dye which will label the nuclei of each cell within the vessel wall. This green fluorescent nucleic acid stain crosses the plasma membrane and intercalates with DNA, thereby labeling the nucleus green upon excitation. This dye allows the total number of cells to be calculated. Segments of rabbit aorta prepared for subsequent in vitro staining, were exposed to the nuclear dye syto 13 (5 × 10−6 M) for 30 min at room temperature. After that, the tissues were incubated at 37 ◦ C with PERM, CY (1, 10 or 100 ␮M) for 30 min and then washed. The control tissues received only ethylic alcohol (at the same % v/v used for treated tissues) as treatment and were kept under the same conditions as the treated tissues. All segments were incubated with EH (1 ␮g/ml) for 15 min and then placed endothelial side-up on a slide. The smooth muscle cells (SMC) were visualized with a Bio-Rad Radiance 2100 laser scanning confocal microscope fitted with an argon-ion laser line (BioRad laboratories, Wisconsin, USA) coupled to a Nikon Optiphot microscope with an air objective X10 (Nikon, NA 0.45). The SMC nuclei, stained with syto 13 were visualized using the argon-ion 488-nm line with a 515-nm barrier filter (pinhole aperture 0.8 mm). The argon-ion was changed to 514-nm line with a 570-nm long pass filter and the images of SMC nuclei, stained with EH, were captured. Single optical slices were acquired from an area below the internal elastic lamina. Treated and control tissues were processed and imaged in parallel with Metamorph Software (Universal Imaging Corporation). Laser settings, photomultiplier gain and offset, pinhole and zoom (2.4) were identical for acquisition of images from treated and control specimens.

2.5. Statistical analysis The experimental data are expressed as mean values ± S.E. and n denotes the number of animals used in each experiment. Statistical analysis was carried

3. Results

EH can be used to localize cells which are permeabilised by the production of free radicals. In brief, cells are impermeable to EH but an increase of free radicals can selectively permeabilize the plasma membrane. Loss of ionic gradients across the membrane allows passage of EH which labels the nuclei. Fig. 1 shows SMC nuclei that are distinguished by their elongated shape. Syto 13 provides a measure of SMC density (green spots) and EH (red spots) identifies the localized biochemical injury within the media caused by incubation with pyrethroids. Labeling with both dyes simultaneously therefore provides a convenient cell viability assay for isolated aortic segments. Fig. 1a shows the SMC of aorta damaged by addition of water, whereas in the Fig. 1b SMC of control tissue appear not damaged (all spots are green). Fig. 1c and d shows the tissues after treatment with 10 ␮M of PERM or CY. Multiple sections of stained aorta were processed to quantify the total viable and non-viable cell numbers expressed as % area occupied by EH stained nuclei (damaged cells) and % area occupied by syto 13 stained nuclei (all cells). The percentage of non-viable to viable cell numbers (% EH stained nuclei) was used as a relative measurement for smooth muscle cells viability. An increase of this percentage indicated a decrease of viability. Table 1 shows a significant increase of % EH stained nuclei in the tissues treated with 10 and 100 ␮M of pyrethroids compared with control tissues. No significant differences can be observed between control and tissues treated with 1 ␮M of pyrethroids. Comparison between tissues treated with low concentrations (10 ␮M) of pyrethroids revealed that the cell viability measured after PERM treatment (5.62 ± 1.74) was lower (P < 0.05) with respect to that obtained after CY treatment (2.58 ± 1.40). A same trend was obtained using high concentrations (100 ␮M) of pyrethroids. 4. Discussion Pyrethroids induce oxidative stress and, as hydrophobic compounds they accumulate in cell membrane and then disturb its structure (Nasuti et al., 2003). Previous studies on erythrocytes showed that their toxicity was linked to different mechanisms, including reactive oxygen species generation and direct decrease of the activity of the antioxidant system (superoxide dismutase, glutathione peroxidase and catalase) (Kale et al., 1999). The consequence of the above effects is an enhanced lipid peroxidation damage to membranes of erythrocytes and then impairment of their functionality (Nasuti et al., 2003; Gabbianelli et al., 2004). Since pyrethroids are involved in reactive oxygen species production and no investigations had been performed on vascular permeability, we studied the influence of CY and PERM treat-

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Fig. 1. Fluorescent photomicrographs of confocal sections of rabbit aorta labeled with syto 13 (5 × 10−6 M, green) and ethidium homodimer (1 ␮g/ml, red). The green spots are the nuclei of the smooth muscle cells. The red spots are the nuclei of damaged smooth muscle cells: (a) rabbit aorta artery damaged by addition of water; (b) rabbit aorta artery of control; (c) rabbit aorta artery treated with permethrin (10 ␮M) for 30 min; (d) rabbit aorta artery treated with cypermethrin (10 ␮M) for 30 min. Image of the sections taken with an LSCM on slide (10× air objective).

ment on rabbit aorta by confocal laser scanning fluorescence microscopy, which allows the study of the blood vessels at the cellular level. This technique produces “optical sections” through semitransparent tissue without the need for cutting thin slices. It eliminates the blur and flare of out-of-focus planes in an object, providing a higher resolution of image. This allows for straightforward measurements at the cellular level with image analysis software, including automated counting of different types of cells as well as determination of several cell parameters such as nuclear shape and area (Daly et al., 2002). Our data showed that treatment of rabbit aortic rings with PERM and CY produced a concentration-dependent damage to smooth muscle cells observed by LSCM.

This study suggested that the free radicals produced by pyrethroids have selectively permeabilised the wall of aorta and then impaired the cell viability of the smooth muscle cells. We could distinguish the different effect of PERM and CY treatment that we could not see with functional studies of sectioned aorta tissues in an organ bath (data not shown). In particular, we observed that at both concentrations (100 and 10 ␮M) the PERM-treatment compromised the smooth muscle cell viability more than CY did. This effect could be related to the differences in structure of the two pyrethroids: PERM is more hydrophobic, so it can go trough the wall of aorta more smoothly than CY and can cause more damaging effects on the SMC layer. In conclusion, PERM seems to induce more oxidative stress on the aorta wall than that CY does.

Table 1 Percentage area occupied by ethidium homodimer and syto 13 stained nuclei in aorta segments untreated, treated with permethrin (PERM) or cypermethrin (CY) (from 100 to 1 ␮M) Group

Dose (␮M)

% area occupied by EH stained nuclei

Control PERM CY PERM CY PERM CY

– 100 100 10 10 1 1

0.19 4.09 2.18 1.48 0.76 0.34 0.29

± ± ± ± ± ± ±

0.12 1.30 0.44 0.43 0.35 0.23 0.28

% area occupied by syto 13 stained nuclei 26.88 27.81 28.80 26.32 29.39 28.40 27.20

± ± ± ± ± ± ±

4.62 4.64 5.15 1.85 4.45 3.12 4.12

% EH stained nuclei 0.71 14.71 7.57 5.62 2.58 1.23 1.10

± ± ± ± ± ± ±

0.40 3.02a,b 0.68a 1.74a,c 1.40a 0.57 0.60

The SMC nuclei were visualized with Bio-Rad 2100 LSCM Laser Sharp and Metamorph software was used for image acquisition and processing, respectively. Data are expressed as mean ± S.D. of 12 tissues (n = 6). a P < 0.05 compared to control tissues. b P < 0.05 compared to CY-treated tissues (100 ␮M). c P < 0.05 compared to CY-treated tissues (10 ␮M).

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Acknowledgements This work was funded by the EC Marie Curie Training Site fellowship contract no. QLRI-CT-2000-60058. We are grateful to TENOVUS Scotland for partial funding of the LSCM. References Bolognesi, C., 2003. Genotoxicology of pesticides: a review of human biomonitoring studies. Mutat. Res. 543, 251–272. Daly, C.J., Gordon, J.F., McGrath, J.C., 1992. The use of fluorescent nuclear dyes for the study of blood vessel structure and function: novel applications of existing techniques. J. Vasc. Res. 29, 41–48. Daly, C.J., McGee, A., Vila, E., Briones, A., Giraldo, J., Arribas, S., Gonzalez, S., Gonzalez, J.M., Somoza, B., Pagatis, S.N., Adler, J., Provost, J.C., Merle, A., Maddison, J., Pederson, J.C., McGrath, J.C., 2002. Analysing the 3D structure of blood vessels using confocal microscopy. Microsc. Microanal. 92, 5–8.

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