The fate of an inhaled cigarette puff in the human respiratory tract.

OBJECTIVE: Cigarette smoking can lead to a host of adverse health effects such as lung and heart disease. Increased lung cancer risk is associated with inhalation of carcinogens present in a puff of smoke. These carcinogenic compounds deposit in the lung at different sites and trigger a cascade of events leading to adverse outcomes. Understanding the site-specific deposition of various smoke constituents will inform the study of respiratory diseases from cigarette smoking. We previously developed a deposition model for inhalation of aerosol from electronic nicotine delivery systems. In this study, the model was modified to simulate inhalation of cigarette smoke consisting of soluble and insoluble tar, nicotine, and cigarette-specific constituents that are known or possible human carcinogens. MATERIALS AND METHODS: The deposition model was further modified to account for nicotine protonation and other cigarette-specific physics-based mechanisms that affect smoke deposition. Model predictions showed a total respiratory tract uptake in the lung for formaldehyde (99%), nicotine (80%), and benzo[a]pyrene (60%). RESULTS: The site of deposition and uptake depended primarily on the constituent's saturation vapor pressure. High vapor pressure constituents such as formaldehyde were preferentially absorbed in the oral cavity and proximal lung regions, while low vapor pressure constituents such as benzo[a]pyrene were deposited in the deep lung regions. Model predictions of exhaled droplet size, droplet retention, nicotine retention, and uptake of aldehydes compared favorably with experimental data. CONCLUSION: The deposition model can be integrated into exposure assessments and other studies that evaluate potential adverse health effects from cigarette smoking.

Role of Calprotectin in Withholding Zinc and Copper from Candida albicans.

The opportunistic fungal pathogen Candida albicans acquires essential metals from the host, yet the host can sequester these micronutrients through a process known as nutritional immunity. How the host withholds metals from C. albicans has been poorly understood; here we examine the role of calprotectin (CP), a transition metal binding protein. When CP depletes bioavailable Zn from the extracellular environment, C. albicans strongly upregulates ZRT1 and PRA1 for Zn import and maintains constant intracellular Zn through numerous cell divisions. We show for the first time that CP can also sequester Cu by binding Cu(II) with subpicomolar affinity. CP blocks fungal acquisition of Cu from serum and induces a Cu starvation stress response involving SOD1 and SOD3 superoxide dismutases. These transcriptional changes are mirrored when C. albicans invades kidneys in a mouse model of disseminated candidiasis, although the responses to Cu and Zn limitations are temporally distinct. The Cu response progresses throughout 72 h, while the Zn response is short-lived. Notably, these stress responses were attenuated in CP null mice, but only at initial stages of infection. Thus, Zn and Cu pools are dynamic at the host-pathogen interface and CP acts early in infection to restrict metal nutrients from C. albicans.

Candida albicans adapts to host copper during infection by swapping metal cofactors for superoxide dismutase.

Copper is both an essential nutrient and potentially toxic metal, and during infection the host can exploit Cu in the control of pathogen growth. Here we describe a clever adaptation to Cu taken by the human fungal pathogen Candida albicans. In laboratory cultures with abundant Cu, C. albicans expresses a Cu-requiring form of superoxide dismutase (Sod1) in the cytosol; but when Cu levels decline, cells switch to an alternative Mn-requiring Sod3. This toggling between Cu- and Mn-SODs is controlled by the Cu-sensing regulator Mac1 and ensures that C. albicans maintains constant SOD activity for cytosolic antioxidant protection despite fluctuating Cu. This response to Cu is initiated during C. albicans invasion of the host where the yeast is exposed to wide variations in Cu. In a murine model of disseminated candidiasis, serum Cu was seen to progressively rise over the course of infection, but this heightened Cu response was not mirrored in host tissue. The kidney that serves as the major site of fungal infection showed an initial rise in Cu, followed by a decline in the metal. C. albicans adjusted its cytosolic SODs accordingly and expressed Cu-Sod1 at early stages of infection, followed by induction of Mn-Sod3 and increases in expression of CTR1 for Cu uptake. Together, these studies demonstrate that fungal infection triggers marked fluctuations in host Cu and C. albicans readily adapts by modulating Cu uptake and by exchanging metal cofactors for antioxidant SODs.

Species-specific activation of Cu/Zn SOD by its CCS copper chaperone in the pathogenic yeast Candida albicans.

Candida albicans is a pathogenic yeast of important public health relevance. Virulence of C. albicans requires a copper and zinc containing superoxide dismutase (SOD1), but the biology of C. albicans SOD1 is poorly understood. To this end, C. albicans SOD1 activation was examined in baker's yeast (Saccharomyces cerevisiae), a eukaryotic expression system that has proven fruitful for the study of SOD1 enzymes from invertebrates, plants, and mammals. In spite of the 80% similarity between S. cerevisiae and C. albicans SOD1 molecules, C. albicans SOD1 is not active in S. cerevisiae. The SOD1 appears incapable of productive interactions with the copper chaperone for SOD1 (CCS1) of S. cerevisiae. C. albicans SOD1 contains a proline at position 144 predicted to dictate dependence on CCS1. By mutation of this proline, C. albicans SOD1 gained activity in S. cerevisiae, and this activity was independent of CCS1. We identified a putative CCS1 gene in C. albicans and created heterozygous and homozygous gene deletions at this locus. Loss of CCS1 resulted in loss of SOD1 activity, consistent with its role as a copper chaperone. C. albicans CCS1 also restored activity to C. albicans SOD1 expressed in S. cerevisiae. C. albicans CCS1 is well adapted for activating its partner SOD1 from C. albicans, but not SOD1 from S. cerevisiae. In spite of the high degree of homology between the SOD1 and CCS1 molecules in these two fungal species, there exists a species-specific barrier in CCS-SOD interactions which may reflect the vastly different lifestyles of the pathogenic versus the noninfectious yeast.

Post-translational modification of Cu/Zn superoxide dismutase under anaerobic conditions.

In eukaryotic organisms, the largely cytosolic copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) enzyme represents a key defense against reactive oxygen toxicity. Although much is known about the biology of this enzyme under aerobic conditions, less is understood regarding the effects of low oxygen levels on Cu/Zn SOD enzymes from diverse organisms. We show here that like bakers' yeast (Saccharomyces cerevisiae), adaptation of the multicellular Caenorhabditis elegans to growth at low oxygen levels involves strong downregulation of its Cu/Zn SOD. Much of this regulation occurs at the post-translational level where CCS-independent activation of Cu/Zn SOD is inhibited. Hypoxia inactivates the endogenous Cu/Zn SOD of C. elegans Cu/Zn SOD as well as a P144 mutant of S. cerevisiae Cu/Zn SOD (herein denoted Sod1p) that is independent of CCS. In our studies of S. cerevisiae Sod1p, we noted a post-translational modification to the inactive enzyme during hypoxia. Analysis of this modification by mass spectrometry revealed phosphorylation at serine 38. Serine 38 represents a putative proline-directed kinase target site located on a solvent-exposed loop that is positioned at one end of the Sod1p ß-barrel, a region immediately adjacent to residues previously shown to influence CCS-dependent activation. Although phosphorylation of serine 38 is minimal when the Sod1p is abundantly active (e.g., high oxygen level), up to 50% of Sod1p can be phosphorylated when CCS activation of the enzyme is blocked, e.g., by hypoxia or low-copper conditions. Serine 38 phosphorylation can be a marker for inactive pools of Sod1p.