- Email: [email protected]
Mechanisms of cytotoxic effects of diclofenac sodium, a prostaglandin synthetase inhibitor, on cultured cells
X ICER Abstracts
Monday, 5:30-790 P.M., Sep 21, 1992 Palazzo Dei Congressi
148
4 8echanisms synthetase
of cytotonic inhibitor,
Nobuo Takahasbil,‘, Kohzaburo Nedical Kedical Diclofenac
Pujikawa’ Research University.
effects
on cultured Hinoru
of diclofenac
sodium,
INVOLVEMENT
a prostaglandin
Takayuki
Kuribara’,
Naomi Taka’ and
sodiun(DFNa)
Department
of Ophthalwlogy”,
is a kind
of prostaglandin synthetase inhibitor cataract surgery and intraocular lens irplantatios to inhibit postoperative inflammation. Recently, it has become known that DPNa eyedrops cause cornea1 epithelial damage. To elucidate the aschanismr of the toxic effects of DPNa, soae experiments including cell youth, transaission electron sicroscopic study and DNA histomaa given by flow cytoaetry vere made using cultured human conjunctival cells. Solutions of DPNa were exposed to the cell sheet for 1 min. DPNa damaged cells dose-dependently. 66%. 50% and 13% of cell growth were visible after exposure to 0.1%. 0.2% and 0.3% DPNa, respectively. 0.1% DPNa exposure resulted in no evident cell danage electron aicroscopically. Effects on the cell cycle were as follows: twelve hours after DPNa exposure, cells of nitotic phase and dead cells increased and 24 hours later, mitotic cells decreased gradually with an increase of more dead cells and 48 hours later, almost all of the mitotic cells disappeared.
and baa been used often in Japan after
and Medical Biophysics, Institute, University of
This study was supported in part by Kabi Pharmacia AB.
149
152
0
EVALUATION OF A TOCICU WRlwKATww OF PARACETAUOL jCSS -121 A) W A n CnYODEL OF CONJUNCTIVAL WWTATION .r-• s1: OpmtreRmdoay DeppNnent, H&i-Dieu de Paris, 1 Place du Parvis NotreDarne. 75181 Pa& ox 04, France 2: Laboratolre Chauvin, BP 1174,34fXtfI Montpellier cx 01, France
REVERSIBLE SUPEROXIDE
EXTINCTION ANION
* Cellular Physiology * Chauvin Research
l
OF
EYE-CUP
B WAVE
Laboratory, Montpellier University, Center, Montpellier, France
ERG
BY
France
Oxidative stress applied to retina is responsible for b wave extinction in vivo as well as in vitro. Most previous experiments have 8hOWn that irreversible retinal damage was induced by oxidative stre8s provoked by agents such as lipop8roxides and hydroxyl radical genemting systems. In the present study, we investigated the acute effect of a superoxide generating system, hypoxanthine + xanthine oxidase (HX + X0) in the electrical activity of the retina in the rabbit eye-cup in vitro model. The posterior portion of the eye was mounted as a membrane in a chamber to separate it into 2 compartments. The retina was perfused with an oxygenated buffered saline. ERG was recorded on flash stimulation with 2 electrodes disposed in both compartments. Pharmacological agents were directly added to the perfusion liquid. Exposure of the retina to HX + X0 induced a rapid decrease in b wave ERG in 5-10 min. Suppmseion of the 02. generating system resulted in an immediate recovery of the signal, suggesting that the responsible factor was site+pecific. Moreover, pretreatment with the glutamate antagonist, APV, partially prevented Oz.‘induced b wave extinction. These results suggest that supemxide anion, which can be generated in the retina under VariOuS circumstances, can affect electrogenesis without inducing irreversible damage of the neuroglial tissue. The protective effect of APV also suggests that Oz.--ERG modifications are related to the release of excitotoxic neurotransmitters.
We inve8tiget8d lhe &l&y of hydroxy rl’aoetanilide (parao8tamol) 1 % eyedrop 8oMion to reduoe oonjunotivai inftamm due to the initative effect of chfodde water in human eyes. ft ~88 a randornlzed, doublemasked, pf6Cebo-cOntrOffed (vehfole) study involving 30 healthy volunteemlhe irritative modei was produced by the application of chloride water (1.4 mg per lfter) on both eye8 for five minutes by the mean8 of swimming oooob8. Each eye reoeived 5 drop8 of p&&o or pamoetamol in the 2 hours preceding the provooative test according to the randomiaerion list. ; and slii lamp ic rating soale of hyperhemia duration and inta&ty, as wefl a8 of mean vafu8 of V.A.R.S. This work, the first trial oondu&d with a topioal formulation of paracetamol in human befogs, 0bjeGtMzes its ability to prevent conjunctival ir?itation. It has to be czmfinned by further studies,
6
IP3 IN THE CCK-INDUCED
We have previously mported that cholecystokbdn, CCK, is a potent constrictor of primate iris spbinete-r muscles. llte pmaent experiments were designed to investigate the role of calcium in the CCK-induced eontmetion of the monkey iris sphincter. Ihe effect on inositol pbmphate pmduetion was also studi& Contraction in the presence and absence of 2.4 mM CM+ was studied using a smotb muscle bath. In the presence of CM+ M(-8s (255 nhi) elicited a response that lasted for at least 50 minutes and suvcd as cont&. Thirty mimstcs after Ca*+removal8ndadditi~ofux)~EDTA~thebuffersolution~respmseofCcK was compared to that of carbachol (lO-5 M). A weak and transiutt response was elicited by both drugs. Arklition of 2.4 mM CaG caused a rapid eontraction of the same order of magnitude as the control. When CCK was added to a Ca*+-free buffer solution and the iris was repeatedl washed before addition of 2.4 mM CaC12, a contraction of approximately x e same size as the control w*s still elicited. To detetmine the involvement of in&to1 phosphatea the iris sphincters wem prelabelled with myo-3H-inositol. Of the paired sphincters (from the same monkey) one served as conuol. The other iris was incubated with CCK-8s. In preliminary experiments a stimulation of IP3 was observed in five of six irises cottlpmd to their cotmols. These results indicate that the CCK-induced contraction of the monkey iris sphincter requires extracellular calcium influx and that CCK stimulates IP3 production in this tissue.
Kanazawa
5
AND
Copenhagen, Lhxmsrk*.
. Institute’,
OF CALCIUM
comcrIoNoFMoNicBYIRIs. of Physiology Uppgala University, Sweden# an August Krogh -T
cells.
Miyakoshi’,
151
7
150
153
9
TEAR PROTEINS AND ENZYMES IN THE CHIMPANZEE 0 9 Van Ha&tg8~, NJ.“. Boddisr. 1) Biochemical Laboratory, The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherhods 2) Zoo, Natura Artis Magi&m, Amsterdam, The Netherlands
DEFICIENT PLASMA ANTIOXIDANT TO PRESENILE CATARACT? * Cellular Physidogy ** Chauvin Research
STATUS
IN SHR: RELATIONSHIP
Laboratory, Montpellier University, Center, Montpellier, France
France
Spontaneously hypertensive rats (SHR) develop lens opacification in the first year of life. Oxidative stress is a major factor implicated in the genesis of cataract in numerous animal models. In the aim of investigating whether presenile SHR cataract can be due to an enhanced oxidative pmcess or to a defect in the antioxidant defenses, we studied the plasma antioxidant status of ‘these rats compared to the parent strain, Wistar Kyoto, at different ages. This activity was measured by determining the plasma capacity to inhibit auto-oxkfation of a brain homogenate. It was found that SHR plasma showed a 2U% decrease in antioxidant activity. This defect is present at the beginning of life, before the appearance of lens opacification, and does not evotve during aging. These results suggest that presenile SHR cataraot can be due to oxidative stress which is also confirmed by the decreased lens glutathione content as a function of age between 10 and 64 weeks.
The present study presents the fust comprehensive investigation of tear fluid in the chimpanzee (Pan ~oglodytcs. Tear fluid was collected during narcosis of 8 chimps. Total protein in chimpanzee tear fluid (8,8 f 0,3 g/l) is not significantly different from total protein of human tear fluid. The values in tear fluid for lysozyme (6.2 f I,5 mg HELM), peroxidase (115 f 18 U/ml) and amylase (3.5 f 0,4 U/ml) in chimps were sigaificastly different from those of human lysozyme 11,8 f 1.6 mg HELM), peroxidase (70 f 5 U/ml) and amylaae (1,0 f 0.2 U/ml). Polyacrylamide gelalectrophoreais of tear tluid of the chimp shows ia comparison with human tear fluid an additional low-molecular protein (< 14 kiloDdtoa).
s.49
Monday, 5:30-790 P.M., Sep 21, 1992 Palazzo Dei Congressi
148
4 8echanisms synthetase
of cytotonic inhibitor,
Nobuo Takahasbil,‘, Kohzaburo Nedical Kedical Diclofenac
Pujikawa’ Research University.
effects
on cultured Hinoru
of diclofenac
sodium,
INVOLVEMENT
a prostaglandin
Takayuki
Kuribara’,
Naomi Taka’ and
sodiun(DFNa)
Department
of Ophthalwlogy”,
is a kind
of prostaglandin synthetase inhibitor cataract surgery and intraocular lens irplantatios to inhibit postoperative inflammation. Recently, it has become known that DPNa eyedrops cause cornea1 epithelial damage. To elucidate the aschanismr of the toxic effects of DPNa, soae experiments including cell youth, transaission electron sicroscopic study and DNA histomaa given by flow cytoaetry vere made using cultured human conjunctival cells. Solutions of DPNa were exposed to the cell sheet for 1 min. DPNa damaged cells dose-dependently. 66%. 50% and 13% of cell growth were visible after exposure to 0.1%. 0.2% and 0.3% DPNa, respectively. 0.1% DPNa exposure resulted in no evident cell danage electron aicroscopically. Effects on the cell cycle were as follows: twelve hours after DPNa exposure, cells of nitotic phase and dead cells increased and 24 hours later, mitotic cells decreased gradually with an increase of more dead cells and 48 hours later, almost all of the mitotic cells disappeared.
and baa been used often in Japan after
and Medical Biophysics, Institute, University of
This study was supported in part by Kabi Pharmacia AB.
149
152
0
EVALUATION OF A TOCICU WRlwKATww OF PARACETAUOL jCSS -121 A) W A n CnYODEL OF CONJUNCTIVAL WWTATION .r-• s1: OpmtreRmdoay DeppNnent, H&i-Dieu de Paris, 1 Place du Parvis NotreDarne. 75181 Pa& ox 04, France 2: Laboratolre Chauvin, BP 1174,34fXtfI Montpellier cx 01, France
REVERSIBLE SUPEROXIDE
EXTINCTION ANION
* Cellular Physiology * Chauvin Research
l
OF
EYE-CUP
B WAVE
Laboratory, Montpellier University, Center, Montpellier, France
ERG
BY
France
Oxidative stress applied to retina is responsible for b wave extinction in vivo as well as in vitro. Most previous experiments have 8hOWn that irreversible retinal damage was induced by oxidative stre8s provoked by agents such as lipop8roxides and hydroxyl radical genemting systems. In the present study, we investigated the acute effect of a superoxide generating system, hypoxanthine + xanthine oxidase (HX + X0) in the electrical activity of the retina in the rabbit eye-cup in vitro model. The posterior portion of the eye was mounted as a membrane in a chamber to separate it into 2 compartments. The retina was perfused with an oxygenated buffered saline. ERG was recorded on flash stimulation with 2 electrodes disposed in both compartments. Pharmacological agents were directly added to the perfusion liquid. Exposure of the retina to HX + X0 induced a rapid decrease in b wave ERG in 5-10 min. Suppmseion of the 02. generating system resulted in an immediate recovery of the signal, suggesting that the responsible factor was site+pecific. Moreover, pretreatment with the glutamate antagonist, APV, partially prevented Oz.‘induced b wave extinction. These results suggest that supemxide anion, which can be generated in the retina under VariOuS circumstances, can affect electrogenesis without inducing irreversible damage of the neuroglial tissue. The protective effect of APV also suggests that Oz.--ERG modifications are related to the release of excitotoxic neurotransmitters.
We inve8tiget8d lhe &l&y of hydroxy rl’aoetanilide (parao8tamol) 1 % eyedrop 8oMion to reduoe oonjunotivai inftamm due to the initative effect of chfodde water in human eyes. ft ~88 a randornlzed, doublemasked, pf6Cebo-cOntrOffed (vehfole) study involving 30 healthy volunteemlhe irritative modei was produced by the application of chloride water (1.4 mg per lfter) on both eye8 for five minutes by the mean8 of swimming oooob8. Each eye reoeived 5 drop8 of p&&o or pamoetamol in the 2 hours preceding the provooative test according to the randomiaerion list. ; and slii lamp ic rating soale of hyperhemia duration and inta&ty, as wefl a8 of mean vafu8 of V.A.R.S. This work, the first trial oondu&d with a topioal formulation of paracetamol in human befogs, 0bjeGtMzes its ability to prevent conjunctival ir?itation. It has to be czmfinned by further studies,
6
IP3 IN THE CCK-INDUCED
We have previously mported that cholecystokbdn, CCK, is a potent constrictor of primate iris spbinete-r muscles. llte pmaent experiments were designed to investigate the role of calcium in the CCK-induced eontmetion of the monkey iris sphincter. Ihe effect on inositol pbmphate pmduetion was also studi& Contraction in the presence and absence of 2.4 mM CM+ was studied using a smotb muscle bath. In the presence of CM+ M(-8s (255 nhi) elicited a response that lasted for at least 50 minutes and suvcd as cont&. Thirty mimstcs after Ca*+removal8ndadditi~ofux)~EDTA~thebuffersolution~respmseofCcK was compared to that of carbachol (lO-5 M). A weak and transiutt response was elicited by both drugs. Arklition of 2.4 mM CaG caused a rapid eontraction of the same order of magnitude as the control. When CCK was added to a Ca*+-free buffer solution and the iris was repeatedl washed before addition of 2.4 mM CaC12, a contraction of approximately x e same size as the control w*s still elicited. To detetmine the involvement of in&to1 phosphatea the iris sphincters wem prelabelled with myo-3H-inositol. Of the paired sphincters (from the same monkey) one served as conuol. The other iris was incubated with CCK-8s. In preliminary experiments a stimulation of IP3 was observed in five of six irises cottlpmd to their cotmols. These results indicate that the CCK-induced contraction of the monkey iris sphincter requires extracellular calcium influx and that CCK stimulates IP3 production in this tissue.
Kanazawa
5
AND
Copenhagen, Lhxmsrk*.
. Institute’,
OF CALCIUM
comcrIoNoFMoNicBYIRIs. of Physiology Uppgala University, Sweden# an August Krogh -T
cells.
Miyakoshi’,
151
7
150
153
9
TEAR PROTEINS AND ENZYMES IN THE CHIMPANZEE 0 9 Van Ha&tg8~, NJ.“. Boddisr. 1) Biochemical Laboratory, The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherhods 2) Zoo, Natura Artis Magi&m, Amsterdam, The Netherlands
DEFICIENT PLASMA ANTIOXIDANT TO PRESENILE CATARACT? * Cellular Physidogy ** Chauvin Research
STATUS
IN SHR: RELATIONSHIP
Laboratory, Montpellier University, Center, Montpellier, France
France
Spontaneously hypertensive rats (SHR) develop lens opacification in the first year of life. Oxidative stress is a major factor implicated in the genesis of cataract in numerous animal models. In the aim of investigating whether presenile SHR cataract can be due to an enhanced oxidative pmcess or to a defect in the antioxidant defenses, we studied the plasma antioxidant status of ‘these rats compared to the parent strain, Wistar Kyoto, at different ages. This activity was measured by determining the plasma capacity to inhibit auto-oxkfation of a brain homogenate. It was found that SHR plasma showed a 2U% decrease in antioxidant activity. This defect is present at the beginning of life, before the appearance of lens opacification, and does not evotve during aging. These results suggest that presenile SHR cataraot can be due to oxidative stress which is also confirmed by the decreased lens glutathione content as a function of age between 10 and 64 weeks.
The present study presents the fust comprehensive investigation of tear fluid in the chimpanzee (Pan ~oglodytcs. Tear fluid was collected during narcosis of 8 chimps. Total protein in chimpanzee tear fluid (8,8 f 0,3 g/l) is not significantly different from total protein of human tear fluid. The values in tear fluid for lysozyme (6.2 f I,5 mg HELM), peroxidase (115 f 18 U/ml) and amylase (3.5 f 0,4 U/ml) in chimps were sigaificastly different from those of human lysozyme 11,8 f 1.6 mg HELM), peroxidase (70 f 5 U/ml) and amylaae (1,0 f 0.2 U/ml). Polyacrylamide gelalectrophoreais of tear tluid of the chimp shows ia comparison with human tear fluid an additional low-molecular protein (< 14 kiloDdtoa).
s.49