- Citado por SciELO
versão impressa ISSN 0716-9868
Rev. chil. anat. v.20 n.2 Temuco 2002
Rev. Chil. Anat., 20(2):169-173, 2002.
ACTIONS OF MEFENAMIC ACID ON PREGNANT ALBINO RATS
EFECTOS DEL ÁCIDO MEFENBÁMICO SOBRE LA RATA PREÑADA
SUMMARY: Misuse and abuse of the non-steroidal anti-inflammatory and analgesic mefenamic acid among pregnant women in developing coutries constitute a matter of medical concern, mainly as a function of the potentially serious side effects of that drug, notably at the digestive system level. Female rats were treated during the entire pregnancy period (from day 0 up to day 20) with 5, 15, or 45 mg/kg of mefenamic acid (MA) once daily, by gavage. Controls received the drug vehicle. We observed that there was a slight yet significant impairment of maternal body weight gain of the animals treated with the two highest doses of MA. Although the drug was proven to exert deleterious effects on kidney and liver metabolic functions, no gross signs of renal or hepatic toxicity were detected in our animals and in their concepts. The digestive effects would be presumably caused by the inhibitory actions of MA on the luminal fluid movement and are accounted for by the observed body weight loss during pregnancy.
KEY WORDS: 1. Mefenamic acid; 2. Toxycology; 3. Rat; 4. Pregnancy.
As inhibitors of prostaglandin synthesis, the non-steroidal antiinflammatory drugs (NSAIDs) all share the property of being good tocolytic agents (Mital et al., 1992). Notwithstanding, their use starting at the 27th week of gestation is contraindicated since they can cross the placental barrier (Mackenzie et al., 1985) and cause adverse effects on the concept. Some of them are primary lung hypertension due to the closure of ductus arteriosus (Menahem, 1991), and oligohydramnion due to the reduction of renal blood flow (Itskovitz et al., 1980). These effects can rise the incidence of perinatal morbimortality (Levin, 1980).
Fenamates constitute a group of NSAIDs to which belong compounds as mefenamic, meclofenamic and flufenamic acids. Although this group is said to have no clear advantages over several other NSAIDs (Roberts II & Morrow, 2001), in particular mefenamic acid (MA) is widely used in Brazil to relieve the pain and discomfort arising from various origins, mainly dysmenorrhea.
Since at a first sight the mechanism of the anti-inflamatory action of fenamates was essentially identical to many other NSAIDs, there would be no explanation for so many women reporting about the higher efficacy of MA to alleviate the premenstrual discomfort and pain. More recently, however, it was stated that fenamates are unique among NSAIDs in inhibiting receptors for PGE2 and PGF2a (Rainsford, 1994). This may represent a major additional site for their action on smooth muscle contractility which is perhaps important in the actions of fenamates in treating primary dysmenorrhea. Besides this, mefenamic acid is the only fenamate to display a central as well as a peripheral analgesic action (Roberts II & Morrow).
In view of its serious side effects, including potentially severe diarrhea associated with steatorrhea and inflammation of the bowel, besides hemolytic anemia, MA is not recommended during pregnancy. Notwithstanding, due to the over-the-counter availability of MA in many developing countries, its use to relieve discomfort and pain of undefinite origin during gestation is a generalized practice.
In an attempt to better understanding fenamate actions on gestation, in this paper we examined the effects of MA administered in a high dosage regimen during the entire period of rat pregnancy.
MATERIAL AND METHOD
Female adult virgin, EPM-1 Wistar rats weighing ca. 200 g, under routine laboratory care, were mated in the proportion of 2 females for every male during 2 h. Pregnancy was verified according to Hamilton & Wolfe (1938) and the finding of spermatozoa in vagina was considered the day zero of pregnancy. Forty pregnant rats were then randomly divided into 4 groups and treated from the 1st up to the 20th day of gestation as follows. C, control group, treated with drug vehicle (21% ethyl alcohol in deionized water containing 1% glycerin, 0.01% Tween® 80 and 0.1% Nipagin M); MA5, MA15 and MA45, groups of animals treated respectively with 5, 15 or 45 mg/kg b.w. of mefenamic acid (Aché Labs.). Drugs and drug vehicle (1 ml/rat) were given once a day at 08:00 h by gavage. Treatments started at the day 0 of pregnancy and extended until the 20th day of gestation. Body weight gain was monitored by weighing the animals at days 0, 7th, 10th and 20th of pregnancy.
At term (20th day) the animals were sacrificed by deep ether anesthesia. Upon laparotomy and uterine horns opening, the sites of implantations and reabsorptions were recorded; the living foetuses and their placentae were carefully removed, loosely passed onto filter paper to remove excess liquid and weighed to the nearest of 0.1 mg. The foetuses were examined for major malformations under a stereo microscope.
Data were analyzed by one-way analysis of variance and the Kruskal-Wallis' or Scheffé's multiple comparisons tests (Sokal & Rohlf, 1969). Whenever appropriate, Friedman's analysis of variance was also used (Siegel, 1975). A 2.01 version GraphPad InStatÔ software was employed for calculations.
RESULTS AND DISCUSSION
Figure 1 shows the linear regressions of body weight increase of pregnant rats throughout the experimental period. Similarly to what occurs in normal rats, it should be noticed that all groups reached their fastest weight gain in the last third of the pregnancy. On the other hand, the groups treated with the higher doses of MA (15 and 45 mg/kg per day) had lower curve slopes than did the controls and the MA 5 group.
| ||Fig. 1. Body weight gain of pregnant rats treated with mefenamic acid (MA). Equations and r2 values of the regression lines are as follows. Control: y = 5.16x + 198.55 (r2 = 0.9211); MA 5 mg/kg: y = 4.93x + 200.61 (r2 = 0.9486); MA 15 mg/kg: y = 3.66x + 196.89 (r2 =0.9047); MA 45 mg/kg: y = 3.43x + 192.88 (r2 = 0.8649). Only the angular coefficients of MA15 and MA45 were significantly lower (P<0.05) than those for the other two groups. Data are mean ± SEM from 10 animals per group.|
Mefenamic acid shares with several other (but not all) non-steroidal antiinflammatory drugs like acetylsalicylic acid, indomethacin and ketoprofen, the ability of directly and dose-dependently scavenging generated nitric oxide radicals (Asanuma et al., 2001). Even though this activity may be viewed as desirable as a means of preventing free radicals-reduction of cell viability and apoptotic nuclear changes mainly in neuronal cells, on the other hand excessive scavenging could lead to inhibition of some physiological effects triggered by inducible nitric oxide synthase activation. This dual effect of MA may help to explain our results with the MA15 and MA45 groups.
In addition, the powerful inhibiting activity of human liver phenol sulfotransferases by non-steroidal antiinflammatory drugs deserves mention. It was observed (Vietri et al., 2000) that mefenamic acid, when administered at therapeutic doses, should impair the hepatic sulfation of those compounds that are substrates of phenol sulfotransferase, with a potency which is 270 fold higher than nimesulide, 475 fold higher than diclofenac and about 8,000 fold higher than ibuprofen. In addition, adult and foetal liver sulfotransferases exhibit different inhibition profiles under the influence of mefenamic acid. Not only foetal enzymes were more suscetible than the adult ones to inhibition by MA, but also this drug behaved as a more potent inhibitor than aspirin on human enzymes (Vietri et al., 2001). On the overall, part of the reduction of body weight gain of rat pregnancy disclosed by the groups MA15 and MA45 would well be attributable to some hepatic toxicity of mefenamic acid.
Another deleterious metabolic action of mefenamic acid which could contribute to the effects observed herein is its ability to uncoupling of mitochondrial oxidative phosphorylation (Masubuchi et al., 2000). Although this effect has been clearly demonstrated in hepatocytes, it conceivably could be considered a general phenomenon leading to more or less significant decreases in cellular ATP contents and the consequent effects thereof will vary depending on the particular cell type or tissue involved.
Although the actions of MA mentioned above could be relevant to the observed maternal impairment of weight gain during pregnancy, its effects on digestive functions are
presumably the most serious in this context. In fact, not only clinical (Isaacs et al., 1987; Tanner & Raghunath, 1988) but also experimental observations (Gullikson et al., 1982) call attention for the potential of MA to causing a coeliac disease-like colonic inflammation. The ability of this agent to produce a laxative response and consequent body weight loss relates to its dose-dependent inhibition of fluid transport outside intestinal lumen.
MA given to rats during short periods (two 3-day periods with an intermediate wash-out period of 4 days) had definite nephrotoxicity manifested as a widespread papillary necrosis (Nguyen et al., 2001). However, especially for renal effects, doses well above those used herein were needed. Being so, no significantly increased mortality or other clinically relevant signs due to nephrotoxicity to MA was to be expected in our rats.
With respect to the gestation products, we observed (Table I) that the treatment with MA did not significantly affect the wet weight of placentae and foetuses. Also, no differences were seen among the groups in relation to the number of implantations, of placentae or of foetuses at term. Similarly, there were no deaths in utero or shortly after uterine opening.
It has been reported that some NSAIDs exhibit a selective inhibitory action on the isoform 2 of cyclooxygenase (COX-2), in contraposition to other drugs which are COX-1 selective or essentially nonselective at all. In fact, COX inhibition by most nonsteroidal antiinflammatory agents conforms to a two-step binding mechanism (Rome & Lands, 1975), of which the first one represents a competitive enzyme inhibition, and the second one involves an alteration of the enzyme-inhibitor complex, probably due to some protein conformational change. This last step is time-dependent (it takes seconds to minutes to occur) and whereas all of the selective COX-2 blocking agents inhibit COX1 and COX2 competitively, they are time-dependent against only COX-2 (that is to say, COX-2 selectivity is a function of the second, time-dependent step) (Copeland et al., 1994).
Such a complex kinetic mechanism of inhibition has made it difficult to compare COX selectivity ratios among nonsteroidal antiinflammatory drugs. According to this approach, mefenamic acid is no longer viewed as a selective COX-2 antagonist (Cryer & Feldman, 1998), but instead as a purely competitive inhibitor of COX-1 and COX-2 (Marnett & Kalgutkar, 1999).
No matter the precise COX isoform is preferentially inhibited, mefenamic acid can cause a highly significant reduction of prostaglandins synthesis. Although our MA-treated pregnant rats presumably had important reductions of compounds derived from COX-1 and COX-2 activation, no apparent significantly deleterious effects other than those on mother weights were detected. Notwithstanding, clinically unsuspected, yet definite effects could be exerted mainly on renal and hepatic compartments, in mothers and/or their concepts, as been recently proven to occur with acetylsalicylic acid (Espiridião et al., 2001), and remain to be investigated.
RESUMEN: En los países del tercer mundo, el uso indiscriminado durante el embarazo del fármaco antiinflamatorio no-esteroide, ácido mefenámico, es un tema de preocupación, fundamentalmente, porque este fármaco posee efectos colaterales potencialmente graves, principalmente, a nivel del aparato digestivo. En este trabajo, ratas hembras fueron tratadas durante toda la preñez (desde el día 0 hasta el día 20 de la gestación) con 5, 15 o 45 mg/kg de ácido mefenámico (AM), una vez al día. Los controles recibieron el vehículo de la droga.
Se observó una discreta, aunque significativa, disminución del ritmo de aumento del peso de las madres tratadas con las dos dosis más altas (15 y 45 mg/kg AM). Si bien hay relatos de que el fármaco puede producir efectos adversos importantes sobre funciones metabólicas hepáticas y renales, no hemos observado señales de toxicidad sobre el hígado o los riñones, tanto de las madres como de sus crías. Los efectos digestivos del AM son inhibición del movimiento del fluído luminal para fuera de la luz del tubo digestivo y por ende, aumento del tránsito intestinal, lo que podría responder por la pérdida de peso corporal materno observada, principalmente, durante el último tercio de la preñez.
PALABRAS CLAVE: 1. Ácido mefenámico; 2. Toxicología; 3. Rata hembra; 4. Preñez.
Asanuma, M.; Nishibayashi-Asanuma, S.; Miyazaki, I.; Kohno, M. & Ogawa, N. Neuroprotective effects of non-steroidal anti-inflammatory drugs by direct scavenging of nitric oxide radicals. J. Neurochem., 76:1895-904, 2001. [ Links ]
Copeland, R.A.; Williams, J.M.; Giannaras, J.; Nurnberg, S.; Covington, M.; Pinto, D.; Pick, S. & Trzaskos, J. M. Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase. Proc. Natl. Acad. Sci. U.S.A. 91:11202-6, 1994. [ Links ]
Cryer, B. & Feldman, M. Cycloocygenase-1 and cyclooxygenase-2 selectivity of widely used nonsteroidal anti-inflammatory drugs. Am. J. Med. 104 :413-21, 1998. [ Links ]
Espiridião, S.; Oliveira-Filho, R. M.; Simões, M. J.; Mamede, J. A. V. & Kulay Jr., L. Liver and kidney ultrastructural changes caused by acetylsalicylic acid treatment during pregnancy in rats. Clin. Exper. Obstet. Gynecol., 2002. (in press). [ Links ]
Gullikson, G. W.; Sender, M. & Bass, P. Laxative-like effects of nonsteroidal anti-inflammatory drugs on intestinal fluid movement and membrane integrity. J. Pharmacol. Exp. Ther., 220:236-42, 1982. [ Links ]
Hamilton, J. B. & Wolfe, J. M. The effect of male hormone substance upon birth and prenatal development in the rat. Anat. Rec., 70:433-40, 1938. [ Links ]
Isaacs, P.E.; Sladen, G. E. & Filipe, I. Mefenamic acid enteropathy. J. Clin. Pathol., 40:1221-7, 1987. [ Links ]
Itskovitz, J.; Abramovici, H. & Brandes, J. M. Oligohydramnion, meconium and perinatal death concurrent with indomethacin treatment in human pregnancy. J. Reprod. Med., 24:137-40, 1980. [ Links ]
Levin, D.L. Effects of inhibition of prostaglandin synthesis on fetal development oxygenation, and the fetal circulation. Semin. Perinatol., 4:35-44, 1980. [ Links ]
Mackenzie, I. Z.; Graf, A. K. & Mitchell, M. D. Prostaglandines in the fetal circulation following maternal ingestion of a prostaglandin synthetase inhibitor during mid-pregnancy. Int. J. Gynecol. Obstet., 23:455-8, 1985. [ Links ]
Marnett, L. J. & Kalgutkar, A. S. Cyclooxygenase 2 inhibitors: discovery, selectivity and the future. Trends Pharmacol. Sci., 20:4659, 1999. [ Links ]
Masubuchi, Y.; Yamada, S. & Horie, T. Possible mechanism of hepatocyte injury induced by diphenylamine and its structurally related nonsteroidal anti-inflammatory drugs. J. Pharmacol. Exp. Ther., 292:9827, 2000. [ Links ]
Menahem, S. Administration of prostaglandin inhibitors to the mother; the potential risk to the fetus and neonate with duct-dependent circulation. Reprod. Fertil. Dev. 3:489-94, 1991. [ Links ]
Mital, P.; Garg, S.; Khuteta, P. P.; Khuteta, S. & Mital, P. Mefenamic acid in prevention of premature labor. J. R. Soc. Health 112:2146, 1992. [ Links ]
Nguyen, T. K.; Obatomi, D. K. & Bach, P. H. Increased urinary uronic acid excretion in experimentally-induced renal papillary necrosis in rats. Ren. Fail., 23:31-42, 2001. [ Links ]
Rainsford, K. D. Non-steroidal anti-inflammatory drugs. In: Dale, M. M.; Foreman, J. C. & Fan, T. P. (Eds.) Textbook of Immunopharmacology. 3. ed. Blackwell, Oxford, 1994. pp 309-19. [ Links ]
Roberts II, L. J. & Morrow, J. D. Analgesic-antipyretic and antiinflammatory agents and drugs employed in the treatment of gout. In: Hardman J.G. & Limbird, L.E. (Eds.) Goodman & Gilman's The Pharmacological Basis of Therapeutics, 10. ed. McGraw-Hill, N.York, 2001. pp 687731. [ Links ]
Rome, L. H. & Lands, W. E. Structural requirements for time-dependent inhibition of prostaglandin biosynthesis by anti-inflammatory drugs. Proc. Natl. Acad. Sci. U.S.A., 72: 48635, 1975. [ Links ]
Siegel, S. Estadística no paramétrica. Trillas, México, 1975, p. 346. [ Links ]
Sokal, R. R. & Rohlf, F. J. Biometry. Freeman Co., San Francisco, 1969. p. 776. [ Links ]
Tanner, A.R. & Raghunath, A.S. Colonic inflammation and nonsteroidal anti-inflammatory drug administration. An assessment of the frequency of the problem. Digestion 41:116-20, 1988. [ Links ]
Vietri, M.; De Santi, C.; Pietrabissa, A.; Mosca, F. & Pacifici, G. M. Inhibition of human liver phenol sulfotransferase by nonsteroidal anti-inflammatory drugs. Eur. J. Clin. Pharmacol., 56:817, 2000. [ Links ]
Vietri, M.; Pietrabissa, A.; Mosca, F.; Rane, A. & Pacifici, G.M. Human adult and foetal liver sulphotransferases: inhibition by mefenamic acid and salicylic acid. Xenobiotica, 31:15361, 2001. [ Links ]
Dirección para correspondencia:
Prof. Dr. Luiz Kulay Junior
Rua Dr. Haberbeck Brandão, 68/71
São Paulo- SP
Recibido : 07-05-2002