Adenosine receptors and stress : studies using methylmercury, caffeine and hypoxia

Brain development is a precisely organized process that can be disturbed by various stress factors present in the diet (e.g. exposure to xenobiotics) as well as insults such as decreased oxygen supply. The consequent adverse changes in nervous system function may not necessarily be apparent until a critical age when neurodevelopmental defects may be unmasked by a subsequent challenge. Adenosine and its receptors (AR) (A1, A2A, A2B and A3) which participate in the brain stress response, are potential targets for neuroprotective drugs. The aim of work presented in this thesis was to investigate a possible role of AR in modulating the effects of developmental alterations caused by exposure to three stressors, the fish pollutant methylmercury (MeHg), the adenosine antagonist caffeine or decreased oxygen supply, before and/or after birth. To address these questions we used both AR ligands and genetically engineered mice with altered expression of AR.

MeHg bioaccumulates in fish, shellfish and sea mammals and is neurotoxic especially to the developing brain since it readily crosses the placenta, blood brain barrier and reaches the breast milk. Exposure of dams to very low levels of MeHg (0.2 mg/l in the drinking water) from gestational day 7 to lactational day 7 caused behavioral alterations in offspring that were sex and age-dependent. A decreased response to psychostimulant amphetamine in 2-month-old males perinatally-treated with MeHg pointed to disturbances in dopaminergic functions. Presence of caffeine, that partially blocks A1R and A2AR, attenuated the locomotor alterations induced by MeHg. The basal and amphetamine-stimulated locomotor activity of A1R knock-out (KO) and A2AR KO mice was relatively unaffected by treatment with MeHg, suggesting that these receptors are involved in the protective effect of perinatal caffeine against MeHg toxicity during gestation and lactation.

Although the health consequences of ordinary caffeine consumption are probably minor, there are lingering concerns about caffeine intake during pregnancy and lactation. Modest maternal caffeine intake (0.3 g/l in the drinking water) in our studies induced long-lasting changes in the mouse offspring, evidenced by an increased motor activity and an amplified response to psychostimulants (cocaine and amphetamine) in adult age, irrespective of sex. Similar alterations were observed in A1R KO mice, as well as mice with partial deletion of A1R gene (A1R heterozygote mice) suggesting that adenosine A1R are involved in the alterations triggered by caffeine exposure during development. Furthermore, if the mother partially lacked A1R the offspring displayed more hyperactivity and responded more strongly to cocaine stimulation as adults than did mice of a WT mother, regardless of their genotype. Our results suggest that perinatal caffeine, by acting on A1R in the mother, is responsible for long-lasting behavioral changes in the offspring.

Characterization of the A3R KO mice with regard to motor activity, response to psychostimulants, and susceptibility to neurotoxic challenge suggested that genetic elimination of this receptor has broad effects in early development. Despite sparse distribution of A3R in neurons, life-long deletion of the A3R caused increased basal motor activity in A3R KO mice, decreased response to drugs that do not primarily act via A3R such as caffeine and cocaine, reduced activity upon natural stimuli (darkness), and greater susceptibility of female mice to MeHg toxicity.

Our in vitro studies focused on identifying the intracellular pathways activated by oxygen deprivation and cobalt chloride (CoCl2) exposure in primary cultures of mouse astrocytes. These cells are particularly important in hypoxia because they immediately activate an array of protective mechanisms that can decrease neuronal injury. Cobalt caused numerous toxic effects in primary glial cells reminiscent of events caused by oxygen deprivation such as activation of HIF-1 pathway with increased expression of HIF-1alpha regulated genes, decreased ATP levels, mitochondrial damage and cell death (apoptosis and necrosis). Thus CoCl2 appears to be a useful tool for mimicking some aspects of oxygen deprivation in astrocytes.

Our preliminary in vitro data suggest that endogenous adenosine and adenosine analogues acting on A1R and A3R exert cytoprotective effects against oxygen deprivation and CoCl2 induced toxicity in primary mouse astrocytes. These observations were supported by our in vivo study using Rice-Vannucci hypoxic ischemia model, where genetic elimination of A1R, A2AR and A3R caused aggravated brain damage in different maturational stages.

Thus, adenosine receptors seem to be important during development. Depending on the subtype they can influence the effect of xenobiotics and protect against ischemic conditions.

List of scientific papers

I. Björklund O, Kahlström J, Salmi P, Ogren SO, Vahter M, Chen JF, Fredholm BB, Daré E (2007). The effects of methylmercury on motor activity are sex- and age-dependent, and modulated by genetic deletion of adenosine receptors and caffeine administration. Toxicology. 241(3): 119-33. Epub 2007 Aug 31.
https://pubmed.ncbi.nlm.nih.gov/17920182

II. Björklund O, Kahlström J, Salmi P, Fredholm BB (2008). Perinatal caffeine, acting on maternal adenosine A1 receptors, causes long-lasting behavioral changes in mouse offspring. [Submitted]

III. Björklund O, Halldner-Henriksson L, Yang JN, Eriksson MT, Jacobson MA, Daré E, Fredholm BB (2008). Decreased response to caffeine and other psychostimulants in A3 adenosine receptor knockout mice. [Submitted]

IV. Karovic O, Tonazzini I, Rebola N, Edström E, Lövdahl C, Fredholm BB, Daré E (2007). Toxic effects of cobalt in primary cultures of mouse astrocytes. Similarities with hypoxia and role of HIF-1alpha. Biochem Pharmacol. 73(5): 694-708. Epub 2006 Nov 17.
https://pubmed.ncbi.nlm.nih.gov/17169330