A COMPARATIVE STUDY OF EFFECTS OF AVERSECTIN C , ABAMECTIN AND IVERMECTIN ON APOPTOSIS OF RAT THYMOCYTES INDUCED BY RADIATION AND DEXAMETHASONE

Korystov Yu. N., V. A. Mosin, V. V. Shaposhnikova, M. Kh. Levitman, A. A. Kudryavtsev, E. B. Kruglyak, T. S. Sterl ina, A. V.Viktorov, V. A. Drinyaev: A Comparative Study of Effects of Aversectin C, Abamectin and Ivermectin on Apoptosis of Rat Thymocytes Induced by Radiation and Dexamethasone. Acta Vet. Brno 1999, 68: 23-29. The effects of avermectins on radiationand dexamethasone-induced apoptosis of rat thymocytes have been studied. In particular, the properties of abamectin (avermectin B1), ivermectin (22,23-dihydroavermectin B1, H2B1) and aversectin C were examined. Aversectin C is a mixture of eight naturally occurring avermectins of the following composition: B1 (42%), B2 (22%), A1 (13%) and A2 (23%). This mixture is currently widely used as active ingredient in veterinary antiparasitic formulations produced by Pharmbiomed Co. Apoptosis was estimated by membrane damage, nuclear pycnosis (or chromatin condensation), DNA fragmentation and percentage of cells with subdiploid DNA. It was shown that aversectin C inhibits thymocyte apoptosis induced by both agents. The IC50 dose was in the range of 0.1-0.3 μg/ml for aversectin C, whereas abamectin and ivermectin produced no effect up to 1 μg/ml. It is suggested that avermectins of the A series are responsible for the high anti-apoptotic activity exhibited by aversectin C. Thus, aversectin C is not only an effective antiparasitic drug but is also capable of exhibiting some other useful activities, e.g. a cytoprotective effect exerted on the mammalian immune cells. Avermectins, antiparasitic drugs, apoptosis, thymocytes, dexamethasone, gamma rays The avermectins are a family of closely related 16-membered macrocyclic lactones produced by the fungus Streptomyces avermitilis. Nearly all the avermectins exhibit a broad spectrum of activity against nematode and arthropode parasites, with the B1a compound being the most potent (Campbel l 1989). Accordingly, it was used as active ingredient in commercial antiparasitic formulations. Until recently, there were basically two types of such avermectin-based active ingredients, i.e. ivermectin (consisting mainly of avermectin H2B1a) and abamectin (predominantly containing avermectin B1a). However, several years ago the Pharmbiomed Co. started manufacturing the veterinary and plant protection formulations based on a new active ingredient aversectin C which is an intrinsic mixture of eight naturally occurring avermectins. Preliminary experiments demonstrated that aversectin C (presumably due to the presence of avermectins of both the Aand B-series) possesses some new useful features compared to ivermectin and abamectin, e.g. with the same high efficacy, the spectrum of activity is broader, level of residues is lower and, hence, the shorter withdrawal periods could be recommended (Simetsky et al. 1994; Berezkina et al. 1996). Furthermore, degradation of aversectin C in soil and water was found to be more effective compared to abamectin and ivermectin (Mosin et al. 1998) and, hence, ecologically aversectin C is safer. ACTA VET. BRNO 1999, 68: 23–29 Address for correspondence: Dr. A.V.Viktorov Pharmbiomed Company, Ltd. 12a Selskokhozyaistvennaya Street, Moscow, 129226, Russia Phone/fax: +7 095 181-2463, 181-0462 E-mail: viktorov@viktorov.msk.ru The study of avermectins as antiparasitic drugs has been focused so far exclusively on the effects of the B1 component and its derivative H2B1 (ivermectin). It was shown that abamectin and ivermectin are lethal for invertebrates and relatively non-toxic for mammals (Campbel l 1989; Cully et al. 1996; Vassi la t is et al. 1997; Huang and Casida 1997). Although the mechanisms of action of avermectins in animals and humans are not completely studied yet, ivermectin is now considered as a drug of choice to treat onchocercosis in humans (Campbel l 1989) and it has been successfully used against some human ectoparasites (Youssef et al. 1995). There are a few reports on changes in the immune system in humans and animals associated with ivermectin treatment of some parasitic diseases. Thus, ivermectin increased the antibody level and antibody-dependent cytotoxicity in animals (Rao et al. 1987). Also, an increase in the T and B cell proliferation rate in vitro in response to different antigens was observed in cells isolated from man treated with ivermectin (Lammie et al. 1992). Quite recently, it was shown that both aversectin C and ivermectin can somewhat reduce in mammals the level of B-lymphocytes and T-helpers, with aversectin C being less immunosuppressive (Sivkov et al. 1998). Here we present a comparative study of the effects of aversectin C, abamectin and ivermectin on the radiationand dexamethasone-induced apoptosis in rat thymocytes. It was found that aversectin C significantly inhibits apoptosis, while abamectin and ivermectin are ineffective. Materials and Methods Reagents Ivermectin and abamectin were supplied by Merck Co. (Germany). HPLC grade solvents were also purchased from Merck. Cell culture media and all other reagents were products of Sigma Co. (St. Louis, MO, USA). Thymocyte isolat ion and t reatment Thymocytes were isolated from male Wistar rats (140-160 g) by a standard procedure (Eidus et al. 1990). The cells were irradiated with 60Co gamma rays at a dose rate of 2.1 Gy/min at room temperature. Dexamethasone (2x10-7 M) was administered to the cells just before incubation at 37 oC. The incubation was carried out in 96-well flat-bottomed plates using 200 μl cell suspension (3×106 cells) per well. Avermectins were added to the cell culture as ethanolic solutions (1-3 μg/ml stock concentrations) immediately after the irradiation procedure or administration of dexamethasone. Ethanol concentration in cultural media did not exceed 0.1% which produced no effects on any parameters studied. Determinat ion of cel l death Cell death was evaluated by the decrease in cell number (cell lysis) and by staining of cells with a 0.04 % solution of the trypan blue dye after 24 hrs incubation (Rao et al. 1987) and expressed as the decrease in cell survival (S): S=Nf/Ni x100%, where Nf is the final concentration of unstained cells and Ni is the initial concentration of unstained cells. Determinat ion of nuclear damage Nuclear damage was evaluated by the chromatin condensation (nuclear pycnosis) and fragmentation of condensed chromatin (nuclear fragmentation) as described in detail elsewhere (Eidus et al. 1994; SchulzeOsthoff et al. 1994). Briefly the damaged nuclei were identified with acetic acid-ethanol (1:3) fixation and Giemsa staining after 6 h incubation. Determinat ion of cel l DNA content The percentage of cells with subdiploid set of DNA (sub-G1 cells) was determined on a flow cytometer (laboratory set-up). Cells were fixed with a cold 70 % ethanol after a 6 hr incubation, transferred into phosphate buffer (pH 7.2), and stained with a Hoechst 33258 dye for 5 min (2 mg/ml). In each series, (20-50) ×103 cells were assayed. The relative amount of cells with DNA content corresponding to different stages of the cell cycle was determined from experimental histograms by using PC. Peak assignment in histograms was made according to Dean and Jef t (1974). Determinat ion of DNA fragmentat ion DNA fragmentation was determined in approximately 1.5 ×107 cells 6 hr after incubation according to procedure (Perot t i et al. 1990). HPLC method Reversed-phase HPLC analyses of composition of aversectin C and purity of abamectin and ivermectin were performed with a Hewlett Packard (1050 series) liquid chromatograph (Germany) equipped with a UV variable 24

The effects of avermectins on radiation-and dexamethasone-induced apoptosis of rat thymocytes have been studied.In particular, the properties of abamectin (avermectin B 1 ), ivermectin (22,23-dihydroavermectin B 1 , H 2 B 1 ) and aversectin C were examined.Aversectin C is a mixture of eight naturally occurring avermectins of the following composition: B 1 (42%), B 2 (22%), A 1 (13%) and A 2 (23%).This mixture is currently widely used as active ingredient in veterinary antiparasitic formulations produced by Pharmbiomed Co. Apoptosis was estimated by membrane damage, nuclear pycnosis (or chromatin condensation), DNA fragmentation and percentage of cells with subdiploid DNA.It was shown that aversectin C inhibits thymocyte apoptosis induced by both agents.The IC 50 dose was in the range of 0.1-0.3µg/ml for aversectin C, whereas abamectin and ivermectin produced no effect up to 1 µg/ml.It is suggested that avermectins of the A series are responsible for the high anti-apoptotic activity exhibited by aversectin C. Thus, aversectin C is not only an effective antiparasitic drug but is also capable of exhibiting some other useful activities, e.g. a cytoprotective effect exerted on the mammalian immune cells.

Avermectins, antiparasitic drugs, apoptosis, thymocytes, dexamethasone, gamma rays
The avermectins are a family of closely related 16-membered macrocyclic lactones produced by the fungus Streptomyces avermitilis.Nearly all the avermectins exhibit a broad spectrum of activity against nematode and arthropode parasites, with the B 1a compound being the most potent (Campbell 1989).Accordingly, it was used as active ingredient in commercial antiparasitic formulations.Until recently, there were basically two types of such avermectin-based active ingredients, i.e. ivermectin (consisting mainly of avermectin H 2 B 1a ) and abamectin (predominantly containing avermectin B 1a ).However, several years ago the Pharmbiomed Co. started manufacturing the veterinary and plant protection formulations based on a new active ingredient -aversectin C which is an intrinsic mixture of eight naturally occurring avermectins.Preliminary experiments demonstrated that aversectin C (presumably due to the presence of avermectins of both the A-and B-series) possesses some new useful features compared to ivermectin and abamectin, e.g. with the same high efficacy, the spectrum of activity is broader, level of residues is lower and, hence, the shorter withdrawal periods could be recommended (Simetsky et al. 1994;Berezkina et al. 1996).Furthermore, degradation of aversectin C in soil and water was found to be more effective compared to abamectin and ivermectin ( M o sin et al. 1998) and, hence, ecologically aversectin C is safer.
The study of avermectins as antiparasitic drugs has been focused so far exclusively on the effects of the B 1 component and its derivative H 2 B 1 (ivermectin).It was shown that abamectin and ivermectin are lethal for invertebrates and relatively non-toxic for mammals (Campbell 1989;Cully et al. 1996;Vassilatis et al. 1997;Huang and Casida 1997).Although the mechanisms of action of avermectins in animals and humans are not completely studied yet, ivermectin is now considered as a drug of choice to treat onchocercosis in humans (Campbell 1989) and it has been successfully used against some human ectoparasites ( Y o ussef et al. 1995).
There are a few reports on changes in the immune system in humans and animals associated with ivermectin treatment of some parasitic diseases.Thus, ivermectin increased the antibody level and antibody-dependent cytotoxicity in animals (Rao et al. 1987).Also, an increase in the T and B cell proliferation rate in vitro in response to different antigens was observed in cells isolated from man treated with ivermectin (Lammie et al. 1992).Quite recently, it was shown that both aversectin C and ivermectin can somewhat reduce in mammals the level of B-lymphocytes and T-helpers, with aversectin C being less immunosuppressive (Sivkov et al. 1998).
Here we present a comparative study of the effects of aversectin C, abamectin and ivermectin on the radiation-and dexamethasone-induced apoptosis in rat thymocytes.It was found that aversectin C significantly inhibits apoptosis, while abamectin and ivermectin are ineffective.

Reagents
Ivermectin and abamectin were supplied by Merck Co. (Germany).HPLC grade solvents were also purchased from Merck.Cell culture media and all other reagents were products of Sigma Co. (St.Louis, MO, USA).

Thymocyte isolation and treatment
Thymocytes were isolated from male Wistar rats (140-160 g) by a standard procedure (Eidus et al. 1990).The cells were irradiated with 60 Co gamma rays at a dose rate of 2.1 Gy/min at room temperature.Dexamethasone (2x10 -7 M) was administered to the cells just before incubation at 37 o C. The incubation was carried out in 96-well flat-bottomed plates using 200 µl cell suspension (3×10 6 cells) per well.Avermectins were added to the cell culture as ethanolic solutions (1-3 µg/ml stock concentrations) immediately after the irradiation procedure or administration of dexamethasone.Ethanol concentration in cultural media did not exceed 0.1% which produced no effects on any parameters studied.

Determination of cell death
Cell death was evaluated by the decrease in cell number (cell lysis) and by staining of cells with a 0.04 % solution of the trypan blue dye after 24 hrs incubation (Rao et al. 1987) and expressed as the decrease in cell survival (S): S=N f /N i x100%, where N f is the final concentration of unstained cells and N i is the initial concentration of unstained cells.

Determination of nuclear damage
Nuclear damage was evaluated by the chromatin condensation (nuclear pycnosis) and fragmentation of condensed chromatin (nuclear fragmentation) as described in detail elsewhere (Eidus et al. 1994;Schulze-Osthoff et al. 1994).Briefly the damaged nuclei were identified with acetic acid-ethanol (1:3) fixation and Giemsa staining after 6 h incubation.

Determination of cell DNA content
The percentage of cells with subdiploid set of DNA (sub-G 1 cells) was determined on a flow cytometer (laboratory set-up).Cells were fixed with a cold 70 % ethanol after a 6 hr incubation, transferred into phosphate buffer (pH 7.2), and stained with a Hoechst 33258 dye for 5 min (2 mg/ml).In each series, (20-50) ×10 3 cells were assayed.The relative amount of cells with DNA content corresponding to different stages of the cell cycle was determined from experimental histograms by using PC.Peak assignment in histograms was made according to Dean and Jeft (1974).

Determination of DNA fragmentation
DNA fragmentation was determined in approximately 1.5 ×10 7 cells 6 hr after incubation according to procedure (Perotti et al. 1990).

HPLC method
Reversed-phase HPLC analyses of composition of aversectin C and purity of abamectin and ivermectin were performed with a Hewlett Packard (1050 series) liquid chromatograph (Germany) equipped with a UV variable wavelength detector (set to 243 nm) operating on-line with PC and using a «Multichrom for Windows» software (Ampersand Co., Russia) to acquire and process chromatographic data.The following chromatographic conditions were used: isocratic elution with methanol/acetonitrile/water (65:20:15, v/v/v/) mixture; analytical Ultrasphere ODS (4.6x250 mm) column with ODS precolumn; 1 ml/min flow rate.Individual avermectin components were identified by their retention times in comparison with authentic commercial standards and published data ( No v a k et al. 1993).

MALDI mass-spectrometry
Mass-spectra of aversectin C were obtained with a MALDI TOF VISION 2000 spectrometer (ThermoBioanalysis, U.K.) operating on-line with PC.The UV laser was set to 337 nm; DHB matrix was used.All avermectins were registered as sodium monovalent molecular cations.Typically 5-20 pmoles of avermectins were used for each MALDI-MS analysis.Under these conditions, practically no fragmentation of the avermectin molecule was observed.

Effect of aversectin C on apoptosis in thymocytes
Curves in Fig. 1 show the effect of aversectin C on cell survival (Fig. 1A), DNA fragmentation (Fig. 1B) and nuclear damage (Fig. 1C) in control, irradiated and dexamethasone treated thymocytes as a function of the drug concentration.At none of the concentrations studied did aversectin C influence these parameters in control cells.However, as concentration of aversectin C was increased, the manifestation of apoptosis in treated cells was progressively diminished: a significant increase in survival rate, decrease in DNA fragmentation and nuclear damage were observed both in irradiated and in dexamethasone-treated thymocytes.For irradiated cells, the addition of aversectin C at concentration of about 0.1 µg/ml resulted in a decrease in DNA fragmentation (Fig. 1B) to approximately a half between control and radiation-induced level (the IC 50 dose).In analogous way, aversectin C caused inhibition of thymocyte apoptosis triggered by  dexamethasone: the IC 50 dose was found to be close to 0.3 µg/ml (Fig. 1A-B).Similar results were obtained for the third parameter of apoptosis studied here, i.e. nuclear damage, when it was shown that aversectin C strongly reduced both dexamethasone-and radiation-induced damage of nuclei in treated cells but produced no change in control thymocytes (Fig. 1C).
The IC 50 value calculated for this criterion was about 0.1-0.2µg/ml and at 1 µg/ml concentration of the drug percentage of nuclei damage was decreased to control level.Effect of aversectin C on distribution of cells with respect to DNA content The flow cytometry data showing the relative amount of cells with different sets of DNA corresponding to different stages of the cell cycle (sub-G 1 , G 1 , S and G 2 ,M) are presented in Fig. 2.After irradiation and dexamethasone treatment, the portion of apoptotic cells with subdiploid DNA (sub-G 1 ) was significantly augmented (Fig 2B and 2C).Administration of aversectin C reduced the percentage of apoptotic cells for both treatments to control levels (Fig. 2D and 2E).The dose dependence of this effect demonstrated that a decrease in percentage of apoptotic cells was accompanied by an increase in the portion of G 1 cells as concentration of aversectin C was increased (data not shown).Effect of aversectin C, abamectin and ivermectin on DNA fragmentation Fig. 3 shows the effect of different active ingredients on DNA fragmentation in control, irradiated and dexamethasone-treated thymocytes.Upon administration of any substance no alteration in the DNA fragmentation pattern in control thymocytes was revealed.However, irradiation and dexamethasone treatments induced a noticeable DNA fragmentation which could be abolished by addition of aversectin C only; abamectin and ivermectin turned out to be ineffective.

Discussion
The present work describes for the first time effects of aversectin C, abamectin and ivermectin on mammalian immunocompetent cells -the rat thymocytes.In particular, the ability of these compounds to affect apoptosis induced by radiation and dexamethasone treatment was examined.Judging by parameters characteristic for apoptosis, i.e. cell survival, DNA fragmentation, nuclear damage and DNA cell distribution pattern, we can conclude that administration of avermectins to control cells produced no detectable changes.However, application of aversectin C to either irradiated or dexamethasone treated thymocytes significantly blocked the development of apoptosis, whereas abamectin and ivermectin were much less effective.It seems only reasonable to suggest that avermectins of the A group, present in aversectin C in tangible amounts (about 36%), should be mainly responsible for the observed activity.
The mechanisms of apoptosis are very complex and diverse with a great number of substances capable of triggering the process and many effector molecules are required for it to develop.Ununexpectedly, apoptosis is still poorly understood.However, the structural and biochemical changes occurring in apoptotic cells are rather consistent and common for many cells and agents and, hence, can be used as reliable criteria.It is well documented that mechanisms of apoptosis triggered in thymocytes by ionizing radiation and dexamethasone are different.In particular, the radiation-but not dexamethasone-induced apoptosis depends on p53 gene (Williams and Smith 1993).Furthermore, the membrane damage, chromatin condensation and DNA fragmentation are known to follow the different metabolic pathways (Eidus et al. 1994;Schulze-Osthoff et al. 1994;Sun et al. 1994).Aversectin C suppresses apoptosis induced by both agents according to all criteria used.This implies that it is capable of blocking apoptosis at a very early stage which is crucial to all subsequent damages of different cell structures characteristic for apoptosis.Unfortunately, basing on our present results no suggestions on how aversectin C exerts its anti-apoptotic effects in thymocytes and what particular sites are targeted in the cell can be made.
Finally, the obtained results allow us to recommend to use the veterinary preparations based on aversectin C, instead of those containing abamectin or ivermectin, in cases when animals experience some immune problems.