Effects of antihistamine drugs on the development of drosophila melanogaster

The effects of antihistamines on organismal development have long been under scrutiny. Loratadine (Ethyl-4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2b]pyridin-11-ylidene)-1-piperidinecarboxylate) It is one of the 2nd generation antihistamine drug active ingredients and is frequently used in the treatment of allergic skin diseases, especially allergic diseases such as atopic eczema, acute rhinitis, hives, eye allergy, hay fever. Various research methods are used to understand whether the factors that living things, especially humans, are exposed to in general, have any effect on living things. Here, these methods are generally determined by considering the type of exposure. However, different methods can also be used depending on the type of cell lines or model organism selected. For substances that can penetrate the living body in different ways, applications can also be made in the form of nutrition, unlike the way of penetration. In the studies conducted, whether these substances have an effect on the developmental stages of the organism is also one of the research topics. At the same time, chronic applications are also made when working with long-term exposure factors. On the other hand, studies also investigate whether these substances have effects across generations. In this study, it was aimed to investigate whether the antihistamine drug active ingredient loratadine, which penetrates into the human body with its use and has long-term use depending on the person, has an effect on the developmental processes and behaviors of F1 and F2 generations of Drosophila melanogaster by providing exposure through the food environment. Media were prepared for Drosophila melanogaster in 250 mL glass bottles. Crosses were made in the culture bottles to collect adult individuals and the stocks to be used for the experiment were created. The stock Drosophila melanogaster were kept in separate culture bottles. The method recommended by the World Health Organization for mosquitoes was applied to determine the threshold value of the active substance loratadine. For this, 1 mL of the active substance prepared at different concentrations was added to 50 mL of standard medium. After 24 hours, the number of individuals that died and survived was noted and the percentage of mortality was calculated. After these procedures, the doses to be used in the experiment were determined as 25, 50 and 100 µg/mL. The experimental setups were constructed by adding 9 mL of Loratadine (LOR) to 4,5 grams of dry Drosophila prepared medium. In order to observe the effect of the active substance on metamorphosis time, 20 females and 20 males were crossed and the day of first emergence of eggs, larvae, pupae and adult flies was recorded. The metamorphosis times of the F1 generation were compared between the control and experimental groups. The adult individuals obtained from the F1 generation were transferred to medium without active substance and the F2 generation was obtained and observations were recorded in the F2 generation. On the other hand, male and female individuals were crossed and larvae of the same age were obtained for substance application to larvae. The test substance was applied to the larvae at the same stage of development and the larvae that did not pupate, pupae that did not become adults and individuals that did not complete their development were counted and noted. The phenotypes of adult individuals were examined and noted under a stereo microscope and removed from the vial. Petri dishes coated with 2% agar were used to measure larval movement. The distance traveled by one larva placed in the center of the petri dish for one minute was measured with millimeter paper. For each concentration, 30 larval movements were observed. To look at the effect of the test substance on negative geotaxis, 20 flies were transferred into a vertical empty glass bottle. The bottle was tapped on a stack of paper, causing the flies to land on the bottom of the bottle. Individuals crossing a height of 10 cm in 10 seconds were then recorded. For the effect on pupal position measurement, the distance traveled by the larvae from the food surface to the pupal area was measured. In addition, the morphology of adult flies was examined under a stereo microscope and the anomalies observed were noted for the F1 and F2 generations. In this study, the effect of the active ingredient loratadine on sex ratio was also examined. Various morphological abnormalities were observed in flies growing on LOR-containing media. These phenotypic abnormalities were curled wingedness, tassel wingedness, spear wingedness and wing atrophy. When the F1 and F2 generations were examined, a statistically significant decrease in the number of normal individuals was found at doses of 50 µg/mL and 100 µg/mL. When the larval to pupal transition rates were examined, it was observed that 100% of the larvae in the control group and 25 µg/mL doses were able to pupate, 94% of the larvae in 50 µg/mL doses were able to pupate, and 86% of the larvae in 100 µg/mL doses were able to pupate. Although 50 µg/mL and 100 µg/mL doses decreased the pupation success of the larvae compared to the other groups, this decrease was not significant. When the pupal-to-adult transition rates were analyzed, it was observed that there was a decrease in the number of adults in the experimental groups compared to the control group. Again, this difference was not significant. When larval movement measurements were analyzed, LOR application at 25 µg/mL and 50 µg/mL doses caused a slowdown in larval movement compared to the control group, but did not cause a statistically significant decrease. The 100 µg/mL dose of LOR caused a significant decrease compared to the control group. In the literature, there are several behavioral toxicity studies investigating the effect of antihistamine active ingredients on behavior in different species. In these studies, there are those who obtained different results from our study as well as those who obtained similar results. Yanai et al. (1999) examined histamine H1 receptor occupancy in young male volunteers and doxepin (H1 antagonist) after antihistamine administration. At the same time, the behavior of H1 receptor knockout mice was observed. As a result of this study, they explained that antihistamines did not show a serious effect in mice, but in humans, cognitive performance deteriorated after taking primary generation antihistamines at recommended doses. In our study, especially the slowing of movements may be due to sedation effect as well as genotoxic effect. Zhdanov et al. (2023) examined the effects of chloropyramine, a first generation antihistamine drug, and loratadine and cetirizine, a second generation drug, on the behavior of adult zebrafish. All three drugs significantly changed the locomotor activity of zebrafish and increased the distance traveled and average speed. In our study, in contrast to this study, the active substance LOR decreased the distance traveled and average speed of Drosophila melanogaster, especially at high doses. In the study with MgO, similar to the results of our study, it was determined that at high doses of magnesium oxide (10 mM), larvae formed pupae closer to the food surface. They explained this effect as the negative effect of magnesium oxide on D. melanogaster energy metabolism. In our study, it may be due to the slowing down of the energy metabolism of the larvae pupating close to the food surface. Considering the doses applied, it was observed that the active substance loratadine negatively affected the development of Drosophila melanogaster at certain doses (50 and 100 µg/mL). When the results were examined, the delay in the metamorphosis process in the F1 generation started to be observed from the 3rd instar, 2nd stage at doses of 50 and 100 µg/mL, while it was observed only during the transition to adult form at 25 µg/mL dose. In the F2 generation, changes were observed only at doses of 50 and 100 µg/mL and during the transition to adult form. Due to these delays in the metamorphosis process, it is thought that LOR may have negatively affected their development due to some effects on developmental genes or the functioning of these genes. The reason why the effect in the F2 generation was less than in the F1 generation may be due to the fact that the parents were exposed to the LOR agent during the experimental process and therefore the F1 generation was more exposed to this agent, while the F2 generation was not directly exposed to the agent. In a study conducted with a similar method, they explained that acetylsalicylic acid and acetaldehyde delayed the metamorphosis process less in the F2 generation of Drosophila melanogaster compared to F1. They stated that the toxic effect of acetylsalicylic acid did not continue in the F2 generation. At the same time, they determined that the metamorphosis period was delayed in the F2 generation of individuals treated with acetaldehyde only. They explained that the reason for this delay was the negative effect of acetaldehyde on the functioning of genes. In another study with antidepressant drugs, they found that sertraline, which only acts at certain doses, prolonged the pupation time of the larvae. They explained that the administered substance prolonged this transition due to the fact that it caused various morphological and physiological disorders by negatively affecting the imaginal discs in the larval body. They also explained that the application of various antidepressant agents may have stimulated the production of serotonin hormone in D. melanogaster, which may cause developmental delay. The delay observed in our study may be due to similar reasons. As a result of another study on Drosophila melanogaster, they explained that magnesium oxide (MgO), a nanoparticle, caused negative effects by causing a decrease in pupation and pupal emergence success. Similar to this study, in our study, it was observed that the LOR active ingredient caused a decrease in pupal formation at doses of 50 µg/mL and 100 µg/mL and in pupal emergence success at all doses. However, this decrease was not statistically significant. Therefore, it cannot be said that the LOR active substance has a significant effect in terms of these criteria. There are also no studies on carcinogenicity and genotoxicity. However, when the reports obtained while the drug was on the market were examined, it was stated that it gave negative results in rats, mice and human peripheral lymphocytes. In other reports reviewed, it was concluded that the active ingredient of the drug had a carcinogenic effect in long-term carcinogenicity studies in liver tumors in mice and rats. In our study, it was concluded that LOR negatively affected the development of Drosophila melanogaster at certain doses. On the other hand, LOR may also have genotoxic effects on Drosophila melanogaster as it causes morphological abnormalities in wing structures. Teratogenic evaluation of two sulfonamide compounds on Drosophila melanogaster. In this study, similar to the results of our study, curled wingedness and spear wingedness abnormalities were detected in individuals developing in the medium containing the substance. As the reason for this, they explained that the number of individuals that completed their development in the medium containing two sulfonamide compounds decreased as the dose applied decreased and this compound had a negative effect on the development of individuals. This negative effect was also reflected in the phenotypic abnormality rate in the wing structure. The abnormalities observed in our study may be due to the changes in the wing structure as it has a negative effect on the normal development of the individual. In conclusion, it can be said that the active substance loratadine used in this study, especially the high doses used in the application, has negative effects on Drosophila melanogaster development and behavior. There is no study in the literature on whether the active substance LOR has any effect on Drosophila melanogaster. Since this study is the first study on the effects of loratadine on the development and behavior of Drosophila melanogaster, it is a source for other studies to be conducted in the future. However, in order to fully reveal the toxic and behavioral effects of loratadine and similar antihistamine drug active ingredients on living organisms, further studies should be carried out with different cell lines and living groups, especially with other model organisms and with different methods.