Δ⁹-Tetrahydrocannabinol disrupts mitochondrial function and cell energetics

Abstract

We have observed rapid and extensive depletion of cellular energy stores by ⌬ 9 -tetrahydrocannabinol (THC) in the pulmonary transformed cell line A549. ATP levels declined dose dependently with an IC50 of 7.5 g/ml of THC after 24-h exposure. Cell death was observed only at concentrations Ͼ10 g/ml. Studies using JC-1, a fluorescent probe for mitochondrial membrane potential, revealed diminished mitochondrial function at THC concentrations as low as 0.5 g/ml. At concentrations of 2.5 or 10 g/ml of THC, a decrease in mitochondrial membrane potential was observed as early as 1 h after THC exposure. Mitochondrial function remained diminished for at least 30 h after THC exposure. Flow cytometry studies on cells exposed to particulate smoke extracts indicate that JC-1 red fluorescence was fivefold lower in cells exposed to marijuana smoke extract relative to cells exposed to tobacco smoke extract.Comparison with a variety of mitochondrial inhibitors demonstrates that THC produced effects similar to that of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, suggesting uncoupling of electron transport. Loss of red JC-1 fluorescence by THC was suppressed by cyclosporin A, suggesting mediation by the mitochondrial permeability transition pore. This disruption of mitochondrial function was sustained for at least 24 h after removal of THC by extensive washing. These results suggest that exposure of the bronchopulmonary epithelium to THC may have important health and physiological consequences. adenosine 5Ј-triphosphate; JC-1; marijuana; flow cytometry; mitochondrial membrane potential RECREATIONAL AND MEDICINAL USE of marijuana remains prevalent despite numerous reports of adverse and toxic consequences (1). The epithelial lining of the lung acts as the primary site exposed to the inhaled components of marijuana smoke, including the high concentrations of cannabinoids associated with the particulate tar fraction. Histopathological alterations in the lung resulting from marijuana smoking include proximal airway inflammation, reserve and goblet cell hyperplasia, squamous cell metaplasia, loss of epithelial microvilli, and cellular atypia (13,35). These changes have been observed by bronchoscopic visualization and by microscopic inspection of human airway mucosal biopsy specimens. The mechanisms underlying these effects, however, are poorly understood and have not been investigated extensively.We previously reported that exposure to marijuana smoke in vitro results in significant oxidative stress and suppression of fas-induced apoptosis (42,43). We have also observed that agents that compromise cell energetics, including antioxidants, enhance the cytotoxic effects produced by ⌬ 9

Clarifying CB2 receptor-dependent and independent effects of THC on human lung epithelial cells

“…The pattern of selective response to marijuana smoke extract relative to tobacco or placebo extracts was similar to the pattern observed previously for loss of Ψ m in A549 cells and in an in vivo smoke inhalation model (Sarafian et al, 2003;Sarafian et al, 2006). This similarity raised the possibility of a causal relationship between THC-mediated disruption of mitochondrial energetics and its effects on chemotaxis.…”

“…In previous studies using lung epithelial cells we observed that THC disrupted mitochondrial membrane potential and lowered ATP levels, an effect also observed with marijuana smoke extract but not with tobacco or placebo marijuana smoke extract (Sarafian et al, 2003;Sarafian et al, 2005). Among the questions raised in these studies was the role of cannabinoid receptors in mediating the effects on cell energetics.…”

“…We have previously shown that THC, alone and in the presence of other smoke components, can regulate gene expression in human lung epithelial cells (Sarafian et al, 2005), disrupt mitochondrial function and cellular energetics (Sarafian et al, 2003) modulate cytokine and immune function (Baldwin et al, 1997;Yuan et al, 2002), and regulate the balance between an apoptotic and necrotic response to injury (Sarafian et al, 2001). The chemotactic activity of lung epithelial cells, the primary focus of this report, is felt to play an important role in lung remodeling, host protection and lung repair (Rennard et al, 2006;Shahabuddin et al, 2006).…”

“…Previous studies revealed that Δ 9 -tetrahydrocannabinol (THC) caused mitochondrial injury in primary lung epithelial cells and in the cell line, A549 (Sarafian et al, 2003;Sarafian et al, 2005). The role of cannabinoid receptors in this injury was unclear, as was the potential impact on cell function.…”

“…We have shown previously that THC and marijuana smoke extract compromise cell energetics in lung epithelial cells in vitro (Sarafian et al, 2003;Sarafian et al, 2005). In both primary human lung epithelial cells and the lung tumor cell line, A549, mitochondrial membrane potential (Ψ m ) is diminished along with cellular ATP level.…”