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Background: Vitamin D deficiency, defined as a serum 25-hydroxyvitamin D [25(OH)D] concentration <20 ng/mL, is correlated with a more atherogenic lipid profile. However, oral vitamin D supplementation does not lower LDL-cholesterol concentrations or raise HDL-cholesterol concentrations. This uncoupling between association and causation may result from a failure of oral vitamin D to mimic the effect of dermally synthesized vitamin D in response to ultraviolet type B (UVB) light. Objective: We tested the hypothesis that, in vitamin D-deficient adults, the replenishment of vitamin D with UVB exposure would lower LDL-cholesterol concentrations compared with the effect of oral vitamin D 3 supplementation. Design: We performed a randomized clinical trial in vitamin D-deficient adults and compared vitamin D replenishment between subjects who received oral vitamin D 3 ( n = 60) and those who received narrow-band UVB exposure ( n = 58) ≤6 mo. Results: There was no difference in the change from baseline LDL-cholesterol concentrations between oral vitamin D 3 and UVB groups (difference in median of oral vitamin D 3 minus that of UVB: 1.5 mg/dL; 95% CI: -5.0, 7.0 mg/dL). There were also no differences within groups or between groups for changes in total or HDL cholesterol or triglycerides. Transcriptional profiling of skin and blood, however, revealed significant upregulation of immune pathway signaling with oral vitamin D 3 but significant downregulation with UVB. Conclusions: Correcting vitamin D deficiency with either oral vitamin D 3 or UVB does not improve the lipid profile. Beyond cholesterol, these 2 modalities of raising 25(OH)D have disparate effects on gene transcription. This trial was registered at clinicaltrials.gov as NCT01688102., (© 2017 American Society for Nutrition.)

Tributyltin (TBT), a toxic and widespread environmental contaminant, has been shown to inhibit natural killer (NK) cell cytotoxic function significantly. Inhibition of NK cell cytotoxic function has the potential to increase viral infections and tumor growth. Upon NK cell binding to lysis-sensitive tumor cells, an intracellular pathway is activated, which generally begins with activation of non-receptor protein tyrosine kinases (PTKs) and ends with mitogen-activated protein kinase (MAPK)-mediated release of lytic granules toward the contacted target cell. In the current studies, we used a cytotoxicity assay to examine how low doses (200nM or lower) of TBT affect cytotoxic function. Additionally, we investigated how low doses of TBT modulate the signaling pathway that dictates lytic granule exocytosis. A 1h exposure to 200, 100, 50 and 25nM TBT significantly decreased cytotoxic function 6d later. We also saw significant activation of p38 and p44/42 by as low as 50nM TBT within ten minutes of exposure. The observed activation of MAPKs, p38 and p44/42, implicated their upstream activators MAPK kinases (MAP2Ks). On examining MAP2Ks, MKK3/6 and MEK1/2, activation was seen within ten minutes. However, when the most upstream signaling molecules in this pathway, non-receptor protein tyrosine kinases (PTKs) such as Syk, ZAP-70, Pyk2 and Src were examined, no significant activation was seen. These data imply that upstream activators of MAP2Ks, MAP2K kinases (MAP3Ks), are activated by TBT exposures and/or that MAP2K phosphatases are being inhibited by TBT. Taken together, these data suggest that TBT-induced activation of MAPKs, p38 and p44/42, is caused by their upstream activators MAP2Ks, MKK3/6 and MEK1/2, respectively.

Natural Killer (NK) cells are a subset of lymphocytes that are capable of killing tumor cells, virally infected cells and antibody coated cells. Tributyltin (TBT) is a toxic chemical used for various industrial purposes such as: slime control in paper mills, disinfection of circulating industrial cooling waters, anti-fouling agents in shower curtains and the preservation of wood. TBT can be found in edible items such as dairy products and fish. This study investigates the mechanism by which TBT exposure decreases the immune function of human NK cells, in vitro. Cytotoxic function, the expression of the cytotoxic proteins (granzyme B and perforin), and cAMP response element binding protein (CREB) phosphorylation were examined. NK cells exposed to 300 nM TBT for 1 h showed no significant decrease in cytotoxic function, levels of granzyme B and perforin, or phosphorylation of CREB. However, mRNA levels for the cytotoxic proteins were significantly decreased. A 24 h exposure to 200 nM TBT caused significant decreases in cytotoxic function, levels of granzyme B and perforin, and levels of granzyme B and perforin mRNA. When NK cells were exposed to 300 nM TBT for 1h followed by a 24 h period in TBT-free media, again there were significant decreases in NK cell cytotoxic function, levels of granzyme B and perforin and their mRNA. A 1h exposure to 300 nM TBT followed by a 48 h period in TBT-free media showed similar changes in cytotoxic function and levels of granzyme B and perforin as seen after 24 h in TBT-free media. Additionally, both of these exposures showed significant decreases in phosphorylation of CREB. These results indicate that TBT exposures can disrupt the transcription of granzyme B and perforin and that this disruption cannot be entirely accounted for by a decrease in phosphorylated CREB (phosphoCREB) levels.

Butyltin (BT) compounds are known for their worldwide contamination. Dibutyltin (DBT) is used as a stabilizer in plastic products, and as a deworming agent in poultry. Poultry products have been shown to contain measurable levels of DBT. Drinking water has also been reported to contain BTs due to leaching from PVC pipes. We, and others, have found measurable levels of DBT in human blood. BTs appear to increase the risk of cancer and other viral infections in exposed individuals. In previous studies we have shown that the tumor killing function of natural killer (NK) lymphocytes was greatly diminished after as little as a 1 h exposure to DBT and the inhibition continued even after removal of the compound. We also showed that there was a significant decrease in NK cell lysis of K562 target cells after an exposure to 1.5 microM DBT for 24 h. This 24 h exposure also decreased the ability of NK cells to bind to tumor cells. Loss of binding function was not seen when NK cells were exposed to 5-10 microM DBT for 1 h. However, NK cells exposed to 5 microM DBT for 1 h and then incubated in DBT-free media for 24, 48, or 96 h, showed a significant loss of tumor-binding function within 24 h. The effects of DBT exposure on seven cell surface molecules that are involved in NK-cell interactions with target cells were investigated. The results indicated that the exposure of NK cells to 1.5 microM DBT for 24 h decreased the expression of CD2, CD11a, CD16, CD11c. There was no decrease in expression of any of the markers studied when NK cells were exposed to 5 microM DBT for 1 h, consistent with the fact that a 1-h exposure had no effect on the ability of NK cells to bind tumor cells. However, when NK cells were exposed to 5 microM DBT for 1 h followed by 24, 48 or 96 h incubations in DBT-free media there was decreased expression of several of the cells surface molecules with the most dramatic decreases being in CD16 and CD56.

Phenyltin (PT) compounds (mono-, di-, and triphenyltins) are used in agricultural and consumer products. They contaminate the environment and have toxic effects on aquatic and terrestrial animals including humans. In an earlier study we demonstrated that PTs (1 micro M, for 1h in vitro exposure) could cause considerable inhibition of the tumor-killing function of human natural killer (NK) cells (as much as 85%). In this study we examined whether cytotoxic function can be recovered after a brief exposure (1h) to PTs. Freshly isolated lymphocytes were exposed to triphenyltin (TPT) or diphenyltin (DPT) for 1h. The compound was then removed and the cells were incubated in PT-free medium for as long as 6 days. The results indicated that exposure to 750nM TPT for 1h caused an approximately 63+/-10% decrease in NK-cytotoxic function. However, if the cells were exposed to 750nM TPT for 1h and then allowed to incubate in TPT-free medium for 24h, there was a 91+/-12% loss of cytotoxic function. NK-cytotoxic function remained inhibited for as long as 6 days after removal of the TPT. A 1-h exposure to as much as 5 micro M DPT caused no loss of NK-cytotoxic function when the cells were tested immediately after the exposure. However, if the cells were allowed to incubate in DPT-free medium for 24h after the 1-h exposure to 5 micro M DPT, cytotoxicity was inhibited by 68+/-29% and this inhibition persisted for at least 6 days. These results indicated that short-term exposure to PTs caused persistent negative effects on human NK-cell function. The persistent effects of PTs are compared to those of the butyltins (BTs).

Triazine (atrazine) and carbamates (maneb, metiram, and ziram) are used as pesticides on a variety of crops around the world. To our knowledge, there have been no studies dealing with the effects of these compounds on human natural killer (NK) cells cytotoxic function. NK cells play a central role in immune defense against tumor development and viral infections. Thus, any agent that interferes with the ability of NK cells to lyse their targets could increase the risk of tumor incidence and/or viral infections. In this study, we examined the effects of atrazine, maneb, metiram, zineb, and ziram on the ability of human NK cells to lyse tumor cells. The compounds were tested in both purified NK cells as well as a cell preparation that contained both T and NK lymphocytes (T/NK cells). Lymphocytes were exposed to the compounds for periods of time ranging from 1 h to 6 days. Exposure of highly purified NK cells to 10 microM atrazine, maneb, or metiram inhibited K562 tumor cell lysis by 63+/-25, 95+/-4, and 50+/-6%, respectively, after a 24 h exposure and by 83+/-21, 70+/-39, and 48+/-41% after a 6-day exposure. Exposure to 2.5 microM ziram for 24 h caused a 99+/-2% decrease in lytic function and at 1 microM for 6 days caused a 96+/-4% decrease. However, when T/NK cells were exposed to atrazine, maneb, or metiram for 24 h only 10 microM atrazine and maneb caused a significant decreases in lytic function (61+/-13 and 38+/-18%) and after 6 days only atrazine was inhibitory (54+/-12%). A 24-h exposure to 2.5-microM ziram caused a 41+/-51% decrease in function, but a 6-day exposure to 1 microM ziram caused no inhibition of lytic function. The results provide evidence of relative toxic potential for the five compounds and the immunomodulatory effects on both T and NK lymphocyte function.

Cytotoxic function of human natural killer (NK) cells is modulated by a variety of cytokines. Interleukins (IL) 2 and 12 are both potent stimulators of NK cell cytotoxic function. Tributyltin (TBT) is used in a variety of consumer products and industrial applications. TBT is found in dairy products, meat, and fish. We and others have shown that there are measurable levels of TBT in human blood. Butyltins appear to increase the risk of cancer and viral infections in exposed individuals. We have demonstrated that the ability of NK cells to kill tumor cells is greatly diminished after a l-h exposure to TBT and that this inhibition persists even after removal of the compound. In the current study we examine the effects of the NK-stimulatory ILs, IL2 and IL12, on the ability of NK cells to recover from the persistent inhibitory effects of a 1-h TBT treatment. Highly purified NK cells (> 95% CD16(+)) or a lymphocyte preparation containing both T lymphocytes and NK cells were treated with 300 nM TBT and then allowed to recover for 24 h, 48 h, 4 days, and 6 days in TBT-free media containing no interleukin, 1000 U/mL IL2, 20 ng/mL IL l2, or a combination of IL2 plus IL12. Tumor killing function was then tested using a radioactive chromium release assay. As seen in our previous studies there is no recovery of NK cell cytotoxic function even after a 6-day recovery period when no interleukin is present in the medium. However, there is significant recovery of NK cytotoxic function when IL2, IL12, or the combination of IL2 plus IL12 is present in the medium during the recovery period., ((c) 2002 Elsevier Science (USA).)

Tributyltin (TBT) was produced in large quantities for use in wood preservation, marine antifouling paints, disinfection of circulating industrial cooling waters and slime control in paper mills. TBT is found in dairy products, meat and fish. We and others have shown that there are measurable levels of TBT in human blood. BTs appear to increase the risk of cancer and viral infections in exposed individuals. In previous studies, we demonstrated that the NK-cytotoxic function of lymphocytes was greatly diminished after a 1-h exposure to 300 nM TBT or a 24-h exposure to 200 nM TBT. Inhibition induced by a 1-h exposure to 300 nM TBT continues even after removal of the compound. There is also decreased ability of NK cells to bind to tumor target cells when they have been exposed to 200 nM TBT for 24 h. This loss of binding function is not seen when NK cells are exposed to 300 nM TBT for 1 h. However, NK cells exposed to 300 nM TBT for 1 h and then incubated in TBT-free media for 24, 48 or 96 h, show a significant loss of tumor-binding function by 96 h. The effects of TBT on cell surface molecules that are crucial to NK cell function is investigated. The data indicate there is a loss of expression of CD16 and CD56 on NK cells exposed to 200 nM TBT for 24 h. There is no decrease in expression of any of the markers studied when NK cells are exposed to 300 nM TBT for 1 h, consistent with the fact that a 1-h exposure has no effect on the ability of NK cells to bind to tumor targets. However, when NK cells are exposed to 300 nM TBT followed by 24, 48 or 96 h incubations in TBT-free media, there is a significant loss of CD16 and CD18 expression after 24 h and of CD16 and CD56 expression after 48 and 96 h.

Cytotoxic function of human natural killer (NK) cells is modulated by a variety of cytokines. Interleukins (IL) 2, 12, 15, and 18 and Interferon gamma (IFNgamma) are potent stimulators of NK cell cytotoxicity. Butyltins (BTs) are used in a variety of consumer products and industrial applications. Dibutyltin (DBT) is found in plastic products, beverages stored in PVC pipes during manufacturing, and poultry products. BTs appear to increase the risk of cancer and viral infections in exposed individuals. Recently, we have demonstrated that the ability of NK cells to kill tumor cells is greatly diminished after a 1-h exposure to dibutyltin. This inhibition of tumor killing function continues even after removal of the compound. There is no significant recovery of NK cytotoxic function even when the cells are allowed to recover for 6 days. In the current study we examine the effects of NK-stimulatory cytokines on the ability of NK cells to recover from the inhibitory effects of a 1-h DBT treatment. Highly purified NK cells (>95% CD16(+)) or a lymphocyte preparation containing both T lymphocytes and NK cells were treated with 5 microM DBT and then allowed to recover for 24 h, 48 h, 4 days, and 6 days in DBT-free medium containing either no cytokine or a maximally stimulatory dose of several NK-stimulatory cytokines. Tumor killing function was tested using a radioactive chromium release assay. As seen in our previous studies there is no recovery of NK cell cytotoxic function even after a 6-day recovery period when no cytokine is present in the medium. However, there is significant recovery of NK cytotoxic function when IL2, IL12, or the combination of IL2 plus IL12 is present in the medium during the recovery period. The other cytokines tested (IL15, IL18, and IFNgamma) were unable to increase the cytotoxicity of DBT-exposed NK cells.

Despite mounting evidence on butyltin (BT) contamination and related immunotoxic effects on wildlife, very little is known about BT-associated immunotoxic effects on humans, particularly the effects on human natural killer (NK) lymphocyte function. Our earlier studies demonstrated that in vitro exposure to environmentally relevant concentrations of BTs negatively affect human NK cells and that there are measurable levels of BTs in human blood. In this study we examined whether the inhibition of NK cell cytotoxic function induced by a brief exposure (1 h) to BTs is reversible when the cells are allowed to recover in BT-free media for up to 6 days. Standard methods were used in chemical preparation, blood sampling, NK cell isolation, and 51-Chromium release assay. The results revealed that exposure to 300 nM TBT for 1 h caused an approximately 65- decrease in NK cytotoxic function, whether the lymphocytes were given as long as a 6-day recovery period or no recovery period. There was no recovery (nor any further loss) of NK cytotoxic function following removal of the compound. Exposure to 5 microM DBT for 1 h showed a 41% decrease in cytotoxic function with 0-h recovery and an 83% decrease after a 24-h recovery period. Thus, not only is there no significant recovery of NK cytotoxic function when the lymphocytes are allowed to incubate in BT-free medium for up to 6 days but there is additional loss of cytotoxic function. The results indicated that short-term exposure to BTs causes persistent negative effects on NK cell ability to kill cancer cells.

Phenyltin (PT) contamination has been reported in water, sediment, and fish. However, the role of PT in weakening human immune function mediated through natural killer (NK) lymphocytes has not been elucidated. In this study, we report the effects of in vitro exposure to triphenyltin (TPT), diphenyltin (DPT), and monophenyltin (MPT) on the function of human NK cells. Exposure to TPT (1 microM, for 1 h) inhibited the tumor killing capacity of NK cells by 85%. Exposure of NK cells to DPT for 1 h (5 microM) and 24h (1.5 microM) reduced tumor lysis by greater than 90%. A 24-h exposure of NK cells to 5 microM MPT reduced tumor lysis by greater than 80%. Assays assessing the ability of NK cells to bind to tumor cells showed that a 24-h pretreatment with TPT, DPT, or MPT reduced NK cell binding to tumor cells by greater than 50%. The toxic potential of the PTs followed the order TPT > DPT > MPT. In comparison with butyltins (BTs), in vitro effects of PTs revealed that these compounds are relatively less toxic to NK cells than BTs. The results of this study provide evidence that phenyltin compounds are immunotoxic to human NK cells under in vitro experimental conditions.

The widespread environmental contamination, bio-accumulation, and toxic effects of butyltins (BTs) in wildlife is well documented, but the role of BTs in debilitating human immune function mediated through natural killer (NK) lymphocytes (a primary immune defense against tumor and virally infected cells) has not been described. In this study, we assessed the effects of in vitro exposure to a range of concentrations (encompassing environmentally relevant concentrations) of MBT, DBT, and TBT on human natural killer lymphocytes obtained from adult male and female donors. TBT inhibited the tumor-killing capacity of NK cells when the NK cells were pretreated in vitro at 200 nM for as little as 1 h. Inhibition of NK cytotoxic function ranged from 40 to greater than 90%. The toxic potential of butyltins followed the order of TBT > DBT > MBT. Conjugation assays revealed that after a 24-h exposure to TBT, there was about a 50% decrease in NK cell binding to tumor cells, indicating alteration of the NK cell receptors for tumor cells. Analysis of whole-blood samples for BTs revealed the presence of detectable concentrations of MBT, DBT, and TBT in all of the donors, indicating possible exposure of NK cells to BTs in the blood. The results of this study provide evidence that butyltin compounds significantly inhibit NK cell function and possible NK cell-mediated immunotoxic potential in humans., (Copyright 1999 Academic Press.)