Additional Table 5.

Recent preclinical and clinical studies evaluating caffeine- and nicotine-induced brain dysfunctions and neurotoxicity

Drug	Species (n, sex)/age	Treatment/duration	Main findings	References
	SH-SY5Y cells	200 mM of ethanol (24 h) followed by 20 mM of caffeine (24 h).	The study found that caffeine potentiates cell death induced by ethanol through mTOR/p70S6K/4E-BP1 inhibition.	Sangaunchom et al., 2020
	PC12 cells	Caffeine (0.5-4 mM) at different time points (24-72 h).	The study found that caffeine induced cell death by inhibiting the Nrf2 pathway.	Chian et al., 2022
	SH-SY5Y cells	Caffeine (10 ng/L, 10 μg/L or 10 mg/L) or carbonyl cyanide 3-chlorophenylhydrazone (5 μΜ) (24 h)	The study found that caffeine altered the expression of genes encoding for serotonin receptor 3A, D2R, monoamine oxidase B, and GABA-transaminase.	Vulin et al., 2022
Caffeine	ICR mouse pups (sample size N.A.); PND 3	Caffeine (a bolus of 80 mg/kg) alone or in combination with midazolam (a bolus of 6 mg/kg), ketamine (a bolus of 40 mg/kg), or fentanyl (a bolus of 40 μg/kg).	The study found that caffeine increased the neurotoxic effects of sedative/anaesthetic drugs.	Cabrera et al., 2017
	C57BL/6J male mice (sample size N.A.); 8-10 weeks old	Caffeine (2×5 mg/kg, i.p.), MDMA (4 × 10 mg/kg, i.p.), or caffeine plus MDMA for 2 days. After 5 days, mice received again the same treatment. In between treatments, mice received caffeine (2×5 mg/kg, i.p.) for 5 days	The study found that caffeine potentiated MDMA- induced neurotoxic effects.	Górska et al., 2018
	C57BL/6J Fcen male mice (sample size N.A.); 25-35 days old	Vehicle, caffeine (3 × 5 mg/kg, i.p.), cocaine (3 × 10 mg/kg, i.p.), caffeine plus cocaine on alternate days for 13 days.	The study found that caffeine plus cocaine treatment induced alterations in thalamocortical physiology.	Rivero-Echeto et al., 2021
	Sprague-Dawley male and female rat pups (sample size N.A.); PND 3	Saline, caffeine citrate (100 mg/kg, s.c.), morphine sulphate (2 mg/kg, s.c.), or caffeine plus morphine for 4 days.	The study found that caffeine plus morphine treatment induced an age- and sex-dependent increase in apoptosis and mitochondrial dysfunction in the brain of neonatal rats.	Kasala et al., 2020
	Pregnant rhesus macaques (gestational day 100-120)	Isoflurane (a bolus of 60 mg/kg, infusion 25 mg/kg/h for 5 h), isoflurane plus caffeine (a bolus of 80 mg/kg for 8 h), or no anaesthesia. Fetal brains were collected 3 h after anaesthesia.	The study found that caffeine augmented the neurotoxic of isoflurane	Noguchi et al., 2018
	C57BL/6 male mice perinatally exposed to nicotine (sample size N.A.); PND 21	Nicotine (100 μg/mL, p.o.), saccharin (2%, p.o.), or water from 3 weeks before mating until weaning of pups. At PND 21, male offspring received methylphenidate (0.75 mg/kg, i.p.) or norbinaltorphimine (20 mg/kg, i.p.).	The study showed that perinatal exposure to nicotine reduced dopamine release in the frontal cortex and caused significant deficits in object-based attention and spatial working memory in the offspring. Such alterations were contrasted by norbinaltorphimine.	Zhang et al., 2021
	C57BL/6 male and female mice perinatally exposed to nicotine (sample size N.A.); PND 20	Nicotine (200 μg/mL plus saccharin 1% in drinking water), saccharin (1% in drinking water), or water from 2 weeks before mating until weaning of pups. At PND 20, offspring were evaluated.	The study found that maternal nicotine exposure dysregulates immune-related gene expression by skewing the polarity of M2 microglia in the hippocampus.	Zhou et al., 2021
Nicotine	Sprague-Dawley male and female rats prenatally exposed to nicotine (sample size N.A.); PSD 32	Prenatal treatment: saline or nicotine (3 mg/kg/day) via osmotic mini-pumps.	The study found that prenatal nicotine treatment sex- dependently altered the development of the corticostriatal dopamine system.	Dwyer et al., 2019
	Humans (n=41 non-smokers, 7 women; n=41 smokers, 6 women); 22-70 years old.	N.A.	The study found that cigarette smoking is associated with thinner cortices in regions implicated in the development of substance abuse or in Alzheimer’s disease.	Durazzo et al., 2018
	Humans (n=362 no smokers, 189 women; n=219 former smokers, 120 women; n=177 current smokers, 57 women); 18-30 years old	N.A.	The study identified a correlation between smoking and reduced grey matter volume in middle-aged individuals.	Elbejjani et al., 2019
	Humans (n=1747, divided in nicotine exposed and non-exposed); 12-27 years old	N.A.	The study found that prenatal exposure to maternal cigarette smoking was associated with lower diffusivity in the corpus callosum.	Björnholm et al., 2020
	Humans (n=1344 non-smokers, n=905 smokers); ageN.A.	N.A.	The study found altered grey matter volumes in chronic smokers.	Yang et al., 2020
	Humans (n=41 children, 24 women, born to non-smoking mothers who were exposed to environmental tobacco smoke); 7 years old or older	N.A.	The study found that environmental tobacco smoke exerts detrimental effects on both structure and function of the thalamus.	Margolis et al., 2021
	Humans (n=35 non-smokers, n=19 smokers, n=31 cannabis and tobacco smokers), 18-37 years old	N.A.	The study revealed that both tobacco and cannabis smoking affect grey matter volume.	Daniju et al., 2022

The articles reported in the table were arranged by model/species (cells, mice, rats, monkeys, humans) and in chronological order. CPu: caudate-putamen nucleus; D2R: dopamine D2 receptor; GABA: γ-aminobutyric acid; i.p.: intraperitoneal injection; mTOR: mammalian target of rapamycin; Nrf2: nuclear factor erythroid-2-related factor; ROS: reactive oxygen species; s.c.: subcutaneous injection; SERT: serotonin transporter.