We predict that ascorbic acid due to its high antioxidant property will help cells to suppress the negative impact of Cd.

All data from the analyses are presented as the mean ± standard deviation (SD). Statistical analyses were performed using the Statistica 10 software (StatSoft Inc., Tulsa, OK, USA). The significance of the obtained differences was analyzed by ANOVA through Duncan and Fisher’s LSD post hoc test.

Homogeneity and normality distribution of experimental data were evaluated by Lavene and Cochran’s test. The statistical significance was set up to p <0.05 *, 0.01 **, and 0.001 *** for the detected statistically significant differences.

As it has been previously reported on various model systems, Cd induces alterations of cellular processes on different levels resulting in cell growth retardation (Yamada et al., 2009; Sandalio et al., 2001; Xu et al., 2019). To evaluate the effect of Cd on the growth ability of S. pombe, cells were subjected to various Cd concentrations supplemented in the culture media. Increasing Cd concentration led to sequential abolishment of the cell growth (Figure 2 red symbols). The effect of ascorbic acid on Cd-mediated cellular impairments was investigated based on previous reports showing that ascorbic acid, as a strong antioxidant compound plays a significant role in alleviating various abiotic stresses (Venkatesh and Park, 2014). Additionally, external compounds such as salicylic acid or selenium, through scavenging reactive oxygen species, can mitigate Cd toxicity (Liu et al., 2016; Lin et al., 2012). Indeed, culture pre-treatment with 10 mM AsA largely improved the growth of cells exposed to Cd (Figure 2 green symbols). Figure 2 depicts the growth intensity of cells treated for 3 hours with various Cd concentrations and the effect of AsA supplementation. AsA markedly enhanced cell growth, additionally acknowledged by the IC₅₀ value. Cells pre-incubated with AsA display a 2.8 times higher IC₅₀ value meaning that in the presence of AsA cells require almost three times the amount of Cd to inhibit cell growth to 50% compared to AsA untreated cells.

Moreover, growth curves of Cd-treated cells expressed as growth intensity ratio of the control show dose and timedependent impact of cadmium and AsA on the cell proliferation (Figure 3).

Growth curves in Figure 3 show dose and time-dependent Cd-derived growth retardations and the significant positive impact of AsA pre-treatment. Hence, the data show that AsA pre-treatment mitigates the negative effect of Cd on cell growth. The statistical significance of the single and the mutual impact of Cd addition and AsA supplementation on the cell growth after 3, 6, and 9 hours of incubation was evaluated by the two-way ANOVA and for more accurate interpretation is depicted in Table 1 (After 3 h, After 6 h, After 9 h).

Another confirmation of the positive effect of AsA against Cd-mediated cell impairment is the protein content measurement that represents cell mass gain. As it is shown in Figure 4, AsA significantly enhanced the amount of protein in the presence of all Cd concentrations but also even in its absence. This implies that AsA widely improves cellular condition resulting in enhanced growth ability represented by elevated mass gain ability. Statistical evaluation by two-way ANOVA of the single and the mutual impact of Cd addition and AsA supplementation on the protein level is shown in Table 1 (Protein). Concomitant with our data a recently published study by Zhang et al. (2019) although on the different model system shows that exogenous application of AsA eliminates the negative effect of Cd on the growth of maize. Moreover, studies with guinea pigs also demonstrate that sufficient doses of ascorbic acid help to restore Cd-induced reduction of immune cells or eliminate nephrotoxicity (Nagyova, Galbavy and Ginter, 1994; Kubová et al., 1993).

To further investigate Cd-mediated toxicity to our model system and its possible elimination by ascorbic acid, the Cd-mediated oxidative stress and antioxidant defense mechanisms have been analyzed. Numerous studies on various model organisms confirmed the cadmium-derived enhancement of ROS production. Wang et al. (2019), Singh et al. (2018), and Li et al. (2019) detected increased free radical (as H₂O₂, O₂^-) formation in the lettuce and tomato seedlings, respectively upon Cd treatment. Moreover, Cd-induced cytotoxicity by excessive ROS was demonstrated by Zhuang et al. (2019) in duck renal tubular epithelial cells, Modareszadeh et al. (2021) in Arabidopsis, and interestingly by Khojastehfar et al. (2015) also in MCF-7 human breast cancer cell line and by Wang et al. (2015) in PC-12 cells. Although Cd does not induce elevation of ROS generation directly (Liu, Qu and Kadiiska, 2009), its impact on excessive free radical formation is unquestionable. Hence, we were interested if S. pombe cell exposure to Cd will cause oxidative stress to the yeast cells. As the end product of the membrane lipid peroxidation is malondialdehyde (MDA), its increasing content indicates an escalation of oxidative stress (Requena et al., 1996). In our experimental conditions, increasing Cd concentration enhanced MDA production in a dose-dependent manner (Figure 5 brown symbols). AsA pre-treatment significantly reduced MDA content (Figure 5 green symbols) suggesting its protective role against Cd-mediated oxidative stress. Culture treatment with AsA did not significantly affect MDA content in the control cells without Cd supplementation, while the addition of all Cd concentrations led to the dramatic increase of MDA amount. However, cells pre-incubated with AsA were largely protected against Cd toxicity. More detailed statistical analyses by two-way ANOVA of the significance of the single and mutual impact of Cd addition and AsA supplementation on MDA content are shown in Table 1 (MDA). Our observation is supported by other studies, revealing that antioxidant additives such as Senna alexandrina extract or salicylic acid or components enhancing the antioxidant capacity of the cell such as selenium or silicon might serve as cells protectors against Cd-mediated oxidative stress (Wang et al., 2020; Liu et al., 2016; Tang et al., 2015; Lin et al., 2012).

To confirm the protective role of AsA through its antioxidant property against Cd toxicity, the activity of catalase (CAT) the antioxidant enzyme responsible for H₂O₂ scavenging, was analyzed. Lower Cd concentration (0.01 mM) led immediately to the enhancement of CAT activity of cells without AsA treatment, while AsA pre-incubation was sufficient to protect cells against Cd-mediated oxidative stress without CAT support. Increasing Cd concentrations in AsA untreated cells at first further enhanced CAT activity, however high Cd concentrations (0.04, and 0.1 mM) consistently interrupted CAT activity, and thus subjected cells to oxidative stress. Strikingly, AsA pre-treatment promoted cellular CAT activity even under exposure to high Cd concentrations leading to extended cell protection (Figure 6). Statistical evaluation by two-way ANOVA of the significance of the single and mutual impact of Cd addition and AsA supplementation on the CAT activity is shown in Table 1 (CAT). Similarly, while CAT activity in Cd-treated roots of Brassica napus has dramatically increased, concomitant AsA supplementation brought CAT activity to a normal state hence triggering decreased sensitivity of the plant towards Cd (Jung et al., 2020). Interestingly, similar to our observations, AsA supplementation to Cd-exposed rats largely improved their antioxidant defense status (Chen et al., 2018).

A presented study reveals that the impact of Cd on the exposed organism, through oxidative stress induction, is highly conserved among species and is evident to a similar extent from yeast to higher organisms such as plants, animals, or humans. Contamination of food, water, and air represents the major pathway of human exposure to Cd including Cd exposure to children through breast milk leads to Cd-mediated adverse effects of human health through oxidative stress induction (Wu et al., 2016; Kippler et al., 2012; Chunhabundit, 2016). Thus, we assume that according to our observation, people living in areas exposed to critical amounts of Cd in the environment that are at high risk of oxidative stress should consider supplementation of their nutrition with AsA.