Teratogenesis Induced by Trimethoprim Sulfamethoxazole in Mice

Swiss albino mice (Mus musculus) were used to conduct the present studies. The animals were placed in steel cages under highly controlled conditions of temperature (27±2°C), humidity (40-45%) and light dark (12h) cycles. A total number of 20 females were used during this experiment which were about 7-9 weeks old having body weight of about 30±2g, 05 females for each dose group and 05 for control. All the protocols of animal husbandry were followed approved by the medical ethics committee of Punjab University, Lahore. For experiment, first objective was to mate male and female under controlled conditions so that exact conception time and day 0 of gestation can be determined. Males and females were kept together for at least 5-7 days before mating would occur, so that estrous cycle of females is regulated. Usually two estrous females were kept with one male in cages. Next morning, these females were examined for presence of vaginal plug, which was considered as confirmation of fertile mating. These females were isolated in separate cages and marked as day “0” of gestation. Trimethoprim sulfamethoxazole (TMP-SMX) is available in the form of tablet with the trade name as “Septra D.S” (GlaxoSmithKline Pakistan). Different dose groups were managed as control, S-I, S-II, and S-III by dissolving TMP-SMX in distilled water in such a way that 0.1ml of dose administered to the mice contained 0.00 μg/g B.W. for control, 4.10 μg/g B.W. for S-I, 8.33 μg/g B.W. for S-II, and 16.66μg/g B.W. for S-III dose groups.

Dams were exposed to TMP-SMX with single dose, daily from 6th-12th day of gestation. The treated mice were kept singly in different cages till day 18 of gestation. On 18th day of gestation, dams were weighed and then anaesthetized with ether. Uteri of dams were dissected out and were placed in 0.9% saline solution after weighing. After counting the implantation sites, the fetuses were recovered from the uteri, fixed in Bouin’s fixative for 48 h and then preserved in 70% alcohol for morphological and morphometric studies (Arshad et al., 2017). Morphological and morphometric studies involved wet weights as well as crown-rump (CR) length measurements of each fetus. Similarly, head circumferences, eye circumferences, forelimb lengths, hindlimb lengths and tail lengths were also subjected to morphometric analyses. The average per litter values were obtained for every parameter in each dose group separately. These values were statistically analyzed by ANOVA using IBM-SPSS 16.

Table I.- Developmental defects produced in 18-days old mice fetuses by TMP/SMX, administered orally to pregnant mice on days 6-12 of gestation.

Morphological observations showed control group as normal in size and form (ears, eyes, limbs and skeleton). There was no resorption observed in control group (Table I). In S-I (4.1µg/g B.W.), as compared to control, the morphological defects i.e. distorted axis, hyperextensions of hind and fore limbs, hemorrhages, and hygromas were observed. The percentage of malformed fetuses in this group was 54.54 with no resorbed fetus (Table I). S-II (8.33µg/g B.W.) showed defects like abdominal cysts, extensions of limbs, low set arms, limb displacements, hemorrhages and hygromas. The malformed and resorbed fetuses percentages were 70.37 and 7.40, respectively (Table I). Defects observed in S-III (16.66µg/g B.W.) were i.e. kyphosis, distorted axis, micromelia, extenions of fore and hind limbs, microcephaly, micro-ophthalmia and intrauterine growth retardation. The percentage of malformations in S-III was 54.16 and of resorptions was 44.99 (Table I).

These results are supported by Crider et al. (2009) study, who studied limb defects occurred in 87 out of 537 pregnancies implants by using this drug. Similarly micro-ophthalmia and neural tube defects were also observed. Among all defects, limb deficiencies were the highest ones in our studies. This is further supported by the findings of Czeizel et al. (2001), that out of 66 pregnancies, there were 10 with neural tube defects and 2 were having limb defects along with other defects.

Morphometric studies showed that there is significant (p <0.05) decrease in the head circumference, eye circumference, fore limb and hind limb sizes, tail sizes, and fetal weights in all treatment groups when compared with the control groups (Table II).

The current morphometric analysis showed that Septra D.S has effects on the fetal growth as our results show the fetuses obtained from the pregnant mice treated with Septra D.S were significantly different from controls in their body weight, crown rump length and other morphometric parameters. These findings are

Table II.- Effects of TMP-SMX on development of 18-days old fetuses recovered from pregnant mice, administered orally with different concentrations on 6-12 days of gestation.

Mean±SEM; Significance: ***, P < 0.001; **, P < 0.01; *, P < 0.05. S-I, 4.1µg/g BW; S-II, 8.3µg/g BW; S-III, 16.66µg/g BW.

in accordance with the findings of Wen et al. (2008) and Hernandez-Diaz et al. (2001). They studied the teratogenic effects in relation to folic acid antagonist’s administration.

There are many resorbtions observed during the present study which are also observed by Matok et al. (2009), who observed abortions in 44 pregnancies and termination of pregnancies due to the use of these folic acid antagonists during the first trimester of pregnancy. Their findings also showed that there are also greater risks of urinary tract and cardiovascular abnormalities induced by Septra D.S.

Hemorrhages on different body parts such as brain, abdomen, foot and sacral regions were also induced by Septra D.S in the fetuses of treated mothers. These observations are similar to Dickerman (1984) that Septran D.S has been implicated as one of the agents responsible for hemorrhagic disease of newborn due to glucose 6 phosphate deficiency. It binds to billirubin in the plasma resulting in coagulation of blood, consequently hemorrhagic spots occurred.

The present study has clearly shown that oral administration of TMP-SMX 4.10, 8.33 and 16.66 µg/g B.W. of mice has potential to induce developmental defects in mice embryos particularly if such exposure occurs at the beginning of organogenesis. Therefore, it is strongly recommended that Septran D.S should be used under highly necessitated conditions in human beings; especially its use should be checked during pregnancy. If its use is unavoidable then folic acid should be used as therapeutic agent.

Authors have declared no conflict of interest.