Determination of Total Folates in Complex Nutritional Drinks and Supplements Using a Tri-Enzyme Microbiological Method and Michaelis-Menten Kinetics.

Background: Recent development of LC methods for the determination of total folates (vitamin B9) in complex matrixes have been hindered by vitamer interconversion and yield variability. The official microbiological method (AOAC Official Methods of Analysis 944.12 and 960.46) uses an end point turbidity reading to determine folate concentration. However, when measuring complex matrixes, shifts are observed in the growth curves of the microorganism and inaccuracies are introduced to this quantification method. Objective/Methods: In addition to the tri-enzyme digestion of the standard microbiological method, we have applied enzyme modeling of the initial velocity of bacterial growth using Michaelis-Menten kinetics to achieve more accurate and reproducible determinations of total folates. Results/Conclusions: Accuracy determined through spike recovery in Infant/Adult Nutritional Drink and a complex vitamin matrix gave values acceptable to AOAC standards of 85-110%. Repeatability of the low mass fraction analyte measured at micrograms per 100 g yielded relative standard deviations <15% for all matrixes tested, including three standard reference materials.

Otitis interna, media, and externa with destruction of the left tympanic bulla and subluxation and septic arthritis of the left temporomandibular joint in an alpaca (Vicugna pacos).

A 1.5-year-old, 37.7 kg, female alpaca was evaluated for a 2-week history of weight loss, left ear droop, and deviation of the rostral mandible to the right. Antemortem radiography and postmortem examination revealed otitis interna, media, and externa, destruction of the left tympanic bulla, and subluxation and septic arthritis of the left temporomandibular joint.

Otite interne, moyenne et externe avec destruction de la bulle tympanique gauche et subluxation et arthrite septique de l'articulation temporomandibulaire gauche chez un alpaga(Vicugna pacos) . Une femelle alpaga âgée de 1,5 an, pesant 37,7 kg, a été évaluée pour une anamnèse de 2 semaines de perte de poids, d'oreille gauche pendante et de déviation de la mandibule rostrale à la droite. Une radiographie avant le décès et une nécropsie ont révélé une otite interne, moyenne et externe, la destruction de la bulle tympanique gauche ainsi qu'une subluxation et l'arthrite septique de l'articulation temporomandibulaire gauche.(Traduit par Isabelle Vallières).

Occupational exposure to formaldehyde and alterations in lymphocyte subsets.

BACKGROUND: Formaldehyde is used in many occupational settings, most notably in manufacturing, health care, and embalming. Formaldehyde has been classified as a human carcinogen, but its mechanism of action remains uncertain. METHODS: We carried out a cross-sectional study of 43 formaldehyde-exposed workers and 51 unexposed age and sex-matched controls in Guangdong, China to study formaldehyde's early biologic effects. To follow up our previous report that the total lymphocyte count was decreased in formaldehyde-exposed workers compared with controls, we evaluated each major lymphocyte subset (i.e., CD4(+) T cells, CD8(+) T cells, natural killer [NK] cells, and B cells) and T cell lymphocyte subset (CD4(+) naïve and memory T cells, CD8(+) naïve and memory T cells, and regulatory T cells). Linear regression of each subset was used to test for differences between exposed workers and controls, adjusting for potential confounders. RESULTS: Total NK cell and T cell counts were about 24% (P = 0.037) and 16% (P = 0.0042) lower, respectively, among exposed workers. Among certain T cell subsets, decreased counts among exposed workers were observed for CD8(+) T cells (P = 0.026), CD8(+) effector memory T cells (P = 0.018), and regulatory T cells (CD4(+) FoxP3(+) : P = 0.04; CD25(+) FoxP3(+) : P = 0.008). CONCLUSIONS: Formaldehyde-exposed workers experienced decreased counts of NK cells, regulatory T cells, and CD8(+) effector memory T cells; however, due to the small sample size; these findings need to be confirmed in larger studies.

Anthelmintic resistance in a herd of alpacas (Vicugna pacos).

A herd of alpacas was examined because of a history of severe endoparasitism, anemia, hypoproteinemia, and weight loss. Resistance of gastrointestinal nematodes to albendazole, fenbendazole, and doramectin was documented. This report suggests that anthelmintic resistance may be an emerging problem in South American camelids in North America.

Résistance aux anthelminthiques dans un troupeau d'alpagas(Vicugna pacos) . Un troupeau d'alpagas a été examiné en raison d'une anamnèse d'endoparasitisme grave, d'anémie, d'hypoprotéinémie et de perte de poids. La résistance des nématodes gastro-intestinaux à l'albendazole, au fenbendazole et à la doramectine a été documentée. Ce rapport suggère que, en Amérique du Nord, la résistance aux anthelminthiques peut être un problème émergent chez les camélidés sud-américains.(Traduit par Isabelle Vallières).

Decreased Numbers of CD4(+) Naive and Effector Memory T Cells, and CD8(+) Naïve T Cells, are Associated with Trichloroethylene Exposure.

Trichloroethylene (TCE) is a volatile chlorinated organic compound that is commonly used as a solvent for lipophilic compounds. Although recognized as an animal carcinogen, TCE's carcinogenic potential in humans is still uncertain. We have carried out a cross-sectional study of 80 workers exposed to TCE and 96 unexposed controls matched on age and sex in Guangdong, China to study TCE's early biologic effects. We previously reported that the total lymphocyte count and each of the major lymphocyte subsets (i.e., CD4(+) T cells, CD8(+) T cells, natural killer cells, and B cells) were decreased in TCE-exposed workers compared to controls, suggesting a selective effect on lymphoid progenitors, and/or lymphocyte survival. To explore which T lymphocyte subsets are affected in the same study population, we investigated the effect of TCE exposure on the numbers of CD4(+) naïve and memory T cells, CD8(+) naïve and memory T cells, and regulatory T cells by FACS analysis. Linear regression of each subset was used to test for differences between exposed workers and controls adjusting for potential confounders. We observed that CD4(+) and CD8(+) naïve T cell counts were about 8% (p = 0.056) and 17% (p = 0.0002) lower, respectively, among exposed workers. CD4(+) effector memory T cell counts were decreased by about 20% among TCE-exposed workers compared to controls (p = 0.001). The selective targeting of TCE on CD8(+) naive and possibly CD4(+) naive T cells, and CD4(+) effector memory T cells, provide further insights into the immunosuppression-related response of human immune cells upon TCE exposure.

Occupational exposure to trichloroethylene is associated with a decline in lymphocyte subsets and soluble CD27 and CD30 markers.

Occupational cohort and case-control studies suggest that trichloroethylene (TCE) exposure may be associated with non-Hodgkin lymphoma (NHL) but findings are not consistent. There is a need for mechanistic studies to evaluate the biologic plausibility of this association. We carried out a cross-sectional molecular epidemiology study of 80 healthy workers that used TCE and 96 comparable unexposed controls in Guangdong, China. Personal exposure measurements were taken over a three-week period before blood collection. Ninety-six percent of workers were exposed to TCE below the current US Occupational Safety and Health Administration Permissible Exposure Limit (100 p.p.m. 8 h time-weighted average), with a mean (SD) of 22.2 (36.0) p.p.m. The total lymphocyte count and each of the major lymphocyte subsets including CD4+ T cells, CD8+ T cells, natural killer (NK) cells and B cells were significantly decreased among the TCE-exposed workers compared with controls (P < 0.05), with evidence of a dose-dependent decline. Further, there was a striking 61% decline in sCD27 plasma level and a 34% decline in sCD30 plasma level among TCE-exposed workers compared with controls. This is the first report that TCE exposure under the current Occupational Safety and Health Administration workplace standard is associated with a decline in all major lymphocyte subsets and sCD27 and sCD30, which play an important role in regulating cellular activity in subsets of T, B and NK cells and are associated with lymphocyte activation. Given that altered immunity is an established risk factor for NHL, these results add to the biologic plausibility that TCE is a possible lymphomagen.

Occupational exposure to formaldehyde, hematotoxicity, and leukemia-specific chromosome changes in cultured myeloid progenitor cells.

There are concerns about the health effects of formaldehyde exposure, including carcinogenicity, in light of elevated indoor air levels in new homes and occupational exposures experienced by workers in health care, embalming, manufacturing, and other industries. Epidemiologic studies suggest that formaldehyde exposure is associated with an increased risk of leukemia. However, the biological plausibility of these findings has been questioned because limited information is available on the ability of formaldehyde to disrupt hematopoietic function. Our objective was to determine if formaldehyde exposure disrupts hematopoietic function and produces leukemia-related chromosome changes in exposed humans. We examined the ability of formaldehyde to disrupt hematopoiesis in a study of 94 workers in China (43 exposed to formaldehyde and 51 frequency-matched controls) by measuring complete blood counts and peripheral stem/progenitor cell colony formation. Further, myeloid progenitor cells, the target for leukemogenesis, were cultured from the workers to quantify the level of leukemia-specific chromosome changes, including monosomy 7 and trisomy 8, in metaphase spreads of these cells. Among exposed workers, peripheral blood cell counts were significantly lowered in a manner consistent with toxic effects on the bone marrow and leukemia-specific chromosome changes were significantly elevated in myeloid blood progenitor cells. These findings suggest that formaldehyde exposure can have an adverse effect on the hematopoietic system and that leukemia induction by formaldehyde is biologically plausible, which heightens concerns about its leukemogenic potential from occupational and environmental exposures.

Depletion of WRN enhances DNA damage in HeLa cells exposed to the benzene metabolite, hydroquinone.

Werner syndrome is a progeroid disorder caused by mutations of the WRN gene. The encoded WRN protein belongs to the family of RecQ helicases that plays a role in the maintenance of genomic stability. Single nucleotide polymorphisms in WRN have been associated with an increased risk for some cancers and were recently linked to benzene hematotoxicity. To further address the role of WRN in benzene toxicity, we employed RNA interference (RNAi) to silence endogenous WRN in HeLa cells and examined the susceptibility of these WRN-depleted cells to the toxic effects of the benzene metabolite hydroquinone. HeLa cells were used as the experimental model because RNAi is highly effective in this system producing almost complete depletion of the target protein. Depletion of WRN led to a decrease in cell proliferation and an enhanced susceptibility to hydroquinone cytotoxicity as revealed by an increase in necrosis. WRN-depleted HeLa cells treated with hydroquinone also displayed an increase in the amount of DNA double-strand breaks as determined by the Comet assay, and an elevated DNA damage response as indicated by the sevenfold induction of gammaH2AX and acetyl-p53 (Lys373 and Lys382) over control levels. Together, these results show that WRN plays an important role in the protection of HeLa cells against the toxicity of the benzene metabolite hydroquinone, specifically in mounting a normal DNA damage response following the induction of DNA double-strand breaks. Further studies in bone marrow-derived stem or progenitor cells are required to confirm our findings in HeLa cells and expand our ability to extrapolate the results to benzene toxicity in humans.

Benzo(a)pyrene and 7,12-dimethylbenz(a)anthrecene differentially affect bone marrow cells of the lymphoid and myeloid lineages.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental contaminants that are carcinogenic and immunosuppressive. Benzo(a)pyrene (BP) and 7,12-dimethylbenz(a)anthracene (DMBA) are two prototypic PAHs known to impair the cell-mediated and humoral immune responses. We have previously shown that, in C57BL/6J mice, total bone marrow (BM) cellularity decreased two-fold following intraperitoneal DMBA treatment but not BP treatment. Here, we have used flow cytometry to demonstrate that BP and DMBA differentially alter the lymphoid and myeloid lineages. Following DMBA treatment, the pro/pre B-lymphocytes (B220(lo)/IgM(-)) and the immature B-lymphocytes (B220(lo)/IgM(+)) significantly decreased, while the mature B-lymphocytes (B220(hi)/IgM(+)) remained unaffected. In contrast, BP treatment decreased the pro/pre B-lymphocytes, and did not affect the immature B-lymphocytes or mature B-lymphocytes. The Gr-1(+) cells of the myeloid lineage were depleted 50% following DMBA treatment and only minimally depleted following BP treatment. Interestingly, the monocytes (7/4(+)1A8(lo)) and neutrophils (7/4(+)1A8(hi)) within this Gr-1(+) population were differentially affected by these PAHs. Monocytes and neutrophils were depleted following DMBA treatment whereas neutrophils decreased and monocytes increased following BP treatment. Although TNFalpha and CYP1B1 are implicated as essential mediators of hypocellularity, the similar induction of TNFalpha mRNA and CYP1B1 mRNA in the BM by BP and DMBA suggests that they are not limiting factors in mediating the different effects of these PAHs. Given that similar amounts of BP and DMBA reach the BM when administered intraperitoneally, their differential effects on the lymphoid and myeloid lineages probably stem from differences in reactive metabolites such as PAH quinones and PAH-dihydrodiol-epoxides.

Induction of CYP1A1 and CYP1B1 in liver and lung by benzo(a)pyrene and 7,12-d imethylbenz(a)anthracene do not affect distribution of polycyclic hydrocarbons to target tissue: role of AhR and CYP1B1 in bone marrow cytotoxicity.

Polycyclic aromatic hydrocarbons (PAHs) (50 mg/kg, i.p.) selectively deplete mouse bone marrow (BM) hematopoietic cells through a process that is dependent on CYP1B1. 7,12-dimethylbenz(a)anthracene (DMBA), which forms greater amounts of dihydrodiol-epoxide-DNA adducts in BM, is much more effective in depleting BM cells than benzo(a)pyrene (BP). BM toxicity by BP is restored in congenic mice expressing a weakly responsive aryl hydrocarbon receptor (AhR(d) replaces AhR(b)). BP strongly induces CYP1A1 around the hepatic vein whereas DMBA produces a weaker diffuse response, paralleling differences in CYP1A1 protein. These responses are absent in AhR(d) mice. BP and DMBA broadly and equally induce CYP1A1 in the lung, while CYP1B1 is induced in bronchial blood vessels. In sternum, CYP1B1 is induced in BM and white fat, whereas CYP1A1 is induced only in brown fat. BP and DMBA levels were similar within blood, lung, and BM and did not rise in AhR(d) mice. In liver, selective decrease of BP was consistent with induced metabolism via CYP1A1, which nevertheless does not determine the blood levels and distribution to BM. Effective delivery of BP to BM is indicated by formation of BP-quinone DNA adducts and the effective induction of CYP1B1. The low formation of BP-dihydrodiol-epoxide-DNA adducts suggests effective AhR induction of BM detoxifying reactions that prevents their formation from dihydrodiols. These findings contrast with the substantial hepatic CYP1A1 contribution for PAHs previously seen for intragastric administration where first pass elimination limits the amount of PAHs reaching the BM.

Bone marrow cytotoxicity of benzo[a]pyrene is dependent on CYP1B1 but is diminished by Ah receptor-mediated induction of CYP1A1 in liver.

We have previously used CYP1B1-null mice to demonstrate that dimethylbenz(a)anthracene (DMBA) requires CYP1B1 for bone marrow (BM) toxicity. Benzo(a)pyrene (BP), a much more potent Ah receptor ligand, shows very different responses that nevertheless depend on CYP1B1. Wild-type (AhR(b)) mice treated with DMBA for 48 h exhibit a large loss in BM cellularity and disruption of marrow structure that is not seen for BP treatment. In congenic mice with a low affinity AhR (AhR(d)), DMBA and BP are equally toxic to the BM whereas AhR(d) x CYP1B1-null mice are fully protected. In situ hybridization demonstrates that CYP1B1 mRNA is constitutively expressed in marrow cells and is induced by PAHs according to their AhR affinity (BP>DMBA), including lower levels in AhR(d) mice. Importantly, expression of CYP1A1 mRNA was undetectable in BM. In wild-type mice, BP treatment leads to a fivefold greater induction of hepatic CYP1A1 than that of DMBA treatment. Neither induction occurs in AhR(d) mice. Thus, hepatic metabolism may prevent BP from reaching the BM, where it can be bioactivated by CYP1B1. Flow cytometric analyses of BM cells showed that there were decreases in granulocytes and lymphocytes following DMBA treatment, but not after BP treatment. These data suggest that there is an inverse relationship between liver metabolism and BM toxicity resulting from limitations on the delivery of PAH to CYP1B1 present in BM, where only very low constitutive levels are needed.