Reply to the opinion paper “The impact of stress on rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide: some theoretical concepts for scientific debate” by Silva et al.

We thank the authors (Silva et al. 2022) for their interest in our paper Social isolation stress facilitates chemically induced oral carcinogenesis (Verza et al. 2021). The answers to their observations regarding our research are below.

First, differently from what has been interpreted by Silva et al. 2022, the rats were not subjected to behavioral tests before oral carcinogenesis induction. Behavioral tests were performed before euthanasia to assess the effects of social isolation stress on depressive-like behavior in rats with oral cancer. Tail suspension test (TST) and forced swimming test (FST) are widely used in basic research and pharmaceutical screening of potential antidepressant treatments in rodents. In both tests, the measurement of immobility behavior is the main parameter to assess the rats’ depressive-like behavior. The immobility behavior is interpreted as a failure of persistence in escape-directed behavior in response to an inescapable stress (Cryan et al. 2005). Regarding the intervals used in the TST and FST, we adopted those recommended by Chermat et al. (1986) and Porsolt et al. (1977), respectively. It is worth to mentioning that these authors described the protocols of each test. We agree with the comments concerning water temperature in the FST and the period of the day that the behavioral tests were performed. These observations are relevant, since rodents are sensitive to changes in temperature, humidity, and circadian rhythms. The information about the water temperature used in the FST was provided in the Materials and methods section of our manuscript “In the first session (pre-test), the rats were forced to swim for 15 min in a narrow cylinder (13 cm in diameter × 24 cm in height) containing water (25 °C ± 2 °C)” (Verza et al. 2021). Then, we described “The second session (test itself) was performed 24 h after the pre-test. In this session, the rats were submitted to the forced swimming test for 5 min under the same pre-test conditions”. Therefore, the same water temperature was used in both phases of the FST. All behavioral tests were performed in the morning, between 08:00 and 10:00 a.m. Regarding the period of social isolation, we affirm in the manuscript that the rats from isolated group were separated from their mothers on the 21st postnatal day and kept isolated until the end of experimental period (Verza et al. 2021), when they reached 240 days of life. This resulted in 219 days of social isolation for the stressed rats. TST and FST were performed 14 and 7 days before euthanasia, respectively.

In mammalian, adulthood is determined by musculoskeletal maturity (Quinn 2005). Rats reach the musculoskeletal maturity at approximately 7–8 months of life (approximately 280 days old) (Quinn 2005; Andreollo et al. 2012). Thus, in our study, unlike what Silva et al. suggested, the rats were adults (and not elderly) at the end of oral carcinogenesis. We agree that different environments can lead to changes in the consumption of water and food as well as body weight. Furthermore, the tumor size and cancer-related pain may also affect these parameters. We recorded the body weight of each animal weekly. Initial (before starting 4NQO treatment) and final (before the animal’s euthanasia) body weight measurements were used to calculate the weight variation throughout the oral carcinogenesis period. In the Results section, we showed that isolated rats had on average a weight loss after carcinogen treatment (− 29.46 ± 15.83 g) compared to grouped rats (54.24 ± 21.28 g) (Verza et al. 2021). The accurate measurement of individual consumption of 4NQO was only possible to be performed for the isolated rats. In the control group, rats were randomly grouped into four animals per cage limiting the analysis of the consumption of the water solution with carcinogen. Furthermore, in the same cages of the control group, there were cancer and leukoplakia (non-cancer) rats, which makes it even more difficult to evaluate the relationship between 4NQO consumption and oral cancer development. Even though, we have calculated the 4NQO intake of both groups using for the control rats (grouped) an average consumption per cage. There were no significant differences between the experimental groups regarding the consumption of carcinogen solution. Previous studies showed no differences in water intake between isolated and grouped rats (Nikolaienko et al. 2020; Cevik et al. 2018).

Regarding the limitation mentioned in the discussion section of our manuscript about the absence of a baseline behavior assessment, we would like to clarify that this measurement would lead to the knowledge of possible changes in depressive-like behavior after oral carcinogenesis within the same experimental group (e.g., pre- versus post-carcinogenesis for the control group or pre- versus post-carcinogenesis for the isolated group). This assessment would be interesting, since, in addition to social isolation, other factors such as 4NQO consumption and the age of animals could affect the rats’ depressive behavior. It is known that social isolation stress can lead to behavioral disorders in human and rodents. Anxiety was cited in the discussion section of our paper only for exemplifying such disorders. Currently, other studies are being carried out by our research team to investigate the brain changes associated with anxiety and depression using the 4NQO-induced oral carcinogenesis model.

We hope that the topic addressed in our study can remain interesting for many researchers, who could provide additional information regarding the effects of social isolation stress on cancer onset and progression.

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