Potential Effects of Boldine on Oxidative Stress, Apoptosis, and Inflammatory Changes Induced by the Methylprednisolone Hepatotoxicity in Male Wistar Rats.

Background: Synthetic glucocorticoid therapeutic agent methylprednisolone (MPL), when used for an extended period of time at high dose, promotes the development of reactive oxygen species (ROS)-induced liver toxicity. This study investigated the role of boldine, a natural antioxidant with anti-apoptotic and anti-inflammatory properties, against MPL-induced hepatoxicity in male Wistar rats. Methods: 120 rats were divided into eight equal groups: G1 (control), G2, 3, and 4 (rats orally administered 5, 10, and 50 mg boldine/kg b.w./day; respectively, for 28 days), G5 (rats intramuscularly injected with 100 mg MPL/kg b.w. only on the last three days), G6, 7, and 8 (rats administered boldine + MPL). After the last MPL injection, rats were sacrificed at intervals of 1, 24, and 48 h. Results: There was a significant decrease in WBCs, RBCs count, and HGB levels, as well as an increase in PLT count, ALT, AST, TG, and LDL levels, and a decrease in HDL level in serum. Oxidative stress markers levels increased at all times, and gene expression of antioxidant enzymes increased at 24h. Immunohistochemical analysis revealed that cytochrome c levels significantly increased after MPL treatment. The COMET assay revealed detectable DNA lesions. There was no immune reactivity of IL-6 expressions as an inflammatory response marker. Conclusions: Oral administration of boldine has a modulatory protective, antioxidant, and anti-apoptotic effect against free radicals.

Cytotoxic and genotoxic effects of e-liquids and their potential associations with nicotine, menthol and phthalate esters.

In this study, we determined DNA damage and chromosome breakage (indicators of genotoxicity) and cell viability (an indicator of cytotoxicity) in human lymphoblastoid TK6 and Chinese hamster ovary (CHO) cells treated with 33 e-liquids using in vitro single cell gel (comet), micronucleus (MN), and trypan blue assays, respectively. We also measured the contents of nicotine, five phthalate esters, and DL-menthol in the e-liquids to examine their effects on DNA damage, chromosome breakage, and cell viability. Our chemical analyses showed that: (1) six e-liquids had nicotine ≥2-fold higher than the manufacture's label claim (2-3.5 mg); (2) both dimethyl- and dibutyl-phthalate levels were >0.1 µg/g, i.e., their threshold limits as additives in cosmetics; and (3) the DL-menthol contents ranged from 0.0003 to 85757.2 µg/g, with those of two e-liquids being >1 mg/g, the threshold limit for trigging sensory irritation. Though all the e-liquids induced DNA damage in TK6 cells, 20 resulted in cell viabilities ≤75%, indicating cytotoxicity, yet the inverse relationship between cell viability and DNA damage (r = -0.628, p = 0.003) might reflect their role as pro-apoptotic and DNA damage inducers. Fifteen e-liquids induced MN% in TK6 cells ≥3-fold that of untreated cells. Some of the increase in %MN might be false due to high cytotoxicity, yet six brands showed acceptable cell viabilities (59-71%), indicating chromosome damage. DNA damage and %MN increased when the TK6 cells were exposed to metabolic activation. The CHO cells were less sensitive to the genotoxic effects of the e-liquids than the TK6 cells. DL-menthol was found to be associated with decreased cell viability and increased DNA damage, even at low levels. We cannot dismiss the presence of other ingredients in e-liquids with cytotoxic/genotoxic properties since out of the 63 different flavors, 47 induced DNA damage (≥3-folds), and 26 reduced cell viability (≤75%) in TK6 cells.

Evaluating the potential genotoxicity of phthalates esters (PAEs) in perfumes using in vitro assays.

We previously reported high levels of phthalate esters (PAEs) added as solvents or fixatives in 47 brands of perfumes. Diethyl phthalate was the most abundant compound (0.232-23,649 ppm), and 83.3% of the perfumes had levels >1 ppm, the threshold limit cited by a Greenpeace investigation. All samples had dimethyl phthalate levels higher than its threshold limit of 0.1 ppm, and 88, 38, and 7% of the perfumes had benzyl butyl phthalate, di(2-ethylhexyl) phthalate, and dibutyl phthalate levels, respectively, above their threshold limits. The role of PAEs as endocrine disruptors has been well documented, but their effect on genotoxic behavior has received little attention. We used in vitro single-cell gel electrophoresis (comet) and micronucleus (MN) assays with human lymphoblastoid TK6 cells to evaluate the genotoxic potency of 42 of the same perfumes and to determine its association with PAEs. All perfumes induced more DNA damage than a negative control (NEG), ≥ 90% of the samples caused more damage than cells treated with the vehicles possibly used in perfume's preparations such as methanol (ME) and ethanol (ET), and 11.6% of the perfumes caused more DNA damage than a positive control (hydrogen peroxide). Chromosome breakage expressed as MN frequency was higher in cells treated with 71.4, 64.3, 57.1, and 4.8% of the perfumes than in NEG, cells treated with ME or ET, and another positive control (x-rays), respectively. The genotoxic responses in the comet and MN assays were not correlated. The comet assay indicated that the damage in TK6 cells treated with five PAEs at concentrations of 0.05 and 0.2 ppm either individually or as a mixture did not differ significantly from the damage in cells treated with the perfumes. Unlike the comet assay, the sensitivity of the MN assay to PAEs was weak at both low and high concentrations, and MN frequencies were generally low. This study demonstrates for the first time the possible contribution of PAEs in perfumes to DNA damage and suggests that their use as solvents or fixatives should be regulated. Other ingredients with mutagenic/genotoxic properties, however, may also have contributed to the DNA damage. Future studies should focus on applying a series of assays that use different cellular models with various endpoints to identify the spectrum of genotoxic mechanisms involved.

Assessing the concentration of phthalate esters (PAEs) and bisphenol A (BPA) and the genotoxic potential of treated wastewater (final effluent) in Saudi Arabia.

Plasticizers such as phthalate esters (PAEs) and bisphenol A (BPA) are highly persistent organic pollutants that tend to bio-accumulate in humans through the soil-plant-animal food chain. Some studies have reported the potential carcinogenic and teratogenic effects in addition to their estrogenic activities. Water resources are scarce in Saudi Arabia, and several wastewater treatment plants (WTPs) have been constructed for agricultural and industrial use. This study was designed to: (1) measure the concentrations of BPA and six PAEs, dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), bis (2-ethylhexyl) phthalate (DEHP) and dioctyl phthalate (DOP), in secondary- and tertiary-treated wastewater collected from five WTPs in three Saudi cities for four to five weeks and (2) test their potential genotoxicity. Three genotoxicological parameters were used: % tail DNA (%T), tail moment (TM) and percentage micronuclei (%MN). Both DBP and DEHP were detected in all treated wastewater samples. DMP, DEP, BBP, DOP, and BPA were found in 83.3, 84.2, 79, 73.7 and 97.4% of the samples, respectively. The levels of DMP (p<0.001), DOP (p<0.001) and BPA (p=0.001) were higher in tertiary- treated wastewater than secondary-treated wastewater, perhaps due to the influence of the molecular weight and polarity of the chemicals. Both weekly sampling frequency and WTP locations significantly affected the variability in our data. Treated wastewater from Wadi Al-Araj was able to induce DNA damage (%T and TM) in human lymphoblastoid TK6 cells that was statistically higher than wastewater from all other WTPs and in untreated TK6 cells (negative control). %MN in samples from both Wadi Al-Araj and Manfouah did not differ statistically but was significantly higher than in the untreated TK6 cells. This study also showed that the samples of tertiary-treated wastewater had a higher genotoxicological potential to induce DNA damage than the samples of secondary-treated wastewater. BPA and some PAEs in the treated wastewater might have the potential to induce genetic damage, despite their low levels. Genotoxicity, however, may also have been due to the presence of other contaminants. Our preliminary findings should be of concern to Saudi agriculture because long-term irrigation with treated wastewater could lead to the accumulation of PAEs and BPA in the soil and ultimately reach the human and animal food chain. WTPs need to remove pollutants more efficiently. Until then, a cautious use of treated wastewater for irrigation is recommended to avoid serious health impacts on local populations.

Identification of a novel peptide derived from the M-phase phosphoprotein 11 (MPP11) leukemic antigen recognized by human CD8+ cytotoxic T lymphocytes.

BACKGROUND AND OBJECTIVES: There is an urgent need for the development of leukemia-targeted immunotherapeutic approaches using defined leukemia-associated antigens that are preferentially expressed by most leukemia subtypes and absent or minimally expressed in vital tissues. M-phase phosphoprotein 11 protein (MPP11) is extensively overexpressed in leukemic cells and therefore is considered an attractive target for leukemia T cell therapy. We sought to identify potential CD8+ cytotoxic T lymphocytes that specifically recognised peptides derived from the MPP11 antigen. METHODS: A computer-based epitope prediction program SYFPEITHI, was used to predict peptides from the MPP11 protein that bind to the most common HLA- A*0201 molecule. Peptide binding capacity to the HLA-A*0201 molecule was measured using the T2 TAP-deficient, HLA-A*0201-positive cell line. Dendritic cells were pulsed with peptides and then used to generate CD8+ cytotoxic T lymphocytes (CTL). The CML leukemic cell line K562-A2.1 naturally expressing the MPP11 antigen and engineered to express the HLA-A*0201 molecule was used as the target cell. RESULTS: We have identified a potential HLA-A*0201 binding epitope (STLCQVEPV) named MPP-4 derived from the MPP11 protein which was used to generate a CTL line. Interestingly, this CTL line specifically recognized peptide-loaded target cells in both ELISPOT and cytotoxic assays. Importantly, this CTL line exerted a cytotoxic effect towards the CML leukemic cell line K562-A2.1. CONCLUSION: This is the first study to describe a novel epitope derived from the MPP11 antigen that has been recognized by human CD8+ CTL.

Enhancement of lytic activity of leukemic cells by CD8+ cytotoxic T lymphocytes generated against a WT1 peptide analogue.

The Wilms tumor antigen 1 (WT1) antigen is over-expressed in human leukemias, making it an attractive target for immunotherapy. Most WT1-specific Cytotoxic T Lymphocytes (CTLs) described so far displayed low avidity, limiting its function. To improve the immunogenicity of CTL epitopes, we replaced the first-amino-acid of two known immunogenic WT1-peptides (126 and 187) with a tyrosine. This modification enhances 126Y analogue-binding ability, triggers significant number of IFN-gamma-producing T cells (P = 0.0003), induces CTL that cross-react with the wild-type peptide, exerts a significant lytic activity against peptide-loaded-targets (P = 0.0006) and HLA-A0201-matched-leukemic cells (P = 0.0014). These data support peptide modification as a feasible approach for the development of a leukemia-vaccine.

The Wilms' tumor antigen is a novel target for human CD4+ regulatory T cells: implications for immunotherapy.

Compelling evidences indicate a key role for regulatory T cells (T(reg)) on the host response to cancer. The Wilms' tumor antigen (WT1) is overexpressed in several human leukemias and thus considered as promising target for development of leukemia vaccine. However, recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of T(regs). We have generated T-cell lines and clones that specifically recognized a WT1-84 (RYFKLSHLQMHSRKH) peptide in an HLA-DRB1*0402-restricted manner. Importantly, they recognized HLA-DRB1*04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a T helper 2 cytokine profile, had a CD4(+)CD25(+)Foxp3(+)GITR(+)CD127(-) T(reg) phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell contact. Priming of alloreactive T cells in the presence of T(regs) strongly inhibited the expansion of natural killer (NK), NK T, and CD8(+) T cells and had an inhibitory effect on NK/NK T cytotoxic activity but not on CD8(+) T cells. Furthermore, priming of T cells with the WT1-126 HLA-A0201-restricted peptide in the presence of T(regs) strongly inhibited the induction of anti-WT1-126 CD8(+) CTL responses as evidenced by both very low cytotoxic activity and IFN-gamma production. Moreover, these T(reg) clones specifically produced granzyme B and selectively induced apoptosis in WT1-84-pulsed autologous antigen-presenting cells but not in apoptotic-resistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 interleukin-5(+)/granzyme B(+)/Foxp3(+) CD4(+) T(regs) in five of eight HLA-DR4(+) acute myeloid leukemia patients. Collectively, our in vitro and in vivo findings strongly suggest important implications for the clinical manipulation of T(regs) in cancer patients.

The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk prognostic factors.

B7-H1 molecule increases the apoptosis of tumor-reactive T lymphocytes and reduces their immunogenicity. Breast cancer is the second most common cause of mortality after lung cancer. Direct evidence linking B7-H1 with cancer has been shown in several malignancies; however, its expression in breast cancer has not been investigated. We used immunohistochemistry to investigate the expression of the B7-H1 molecule in 44 breast cancer specimens and to study its correlation with patients' clinicopathological parameters. The expression of B7-H1 was shown in 22 of 44 patients and was not restricted to the tumor epithelium (15 of 44, 34% in tumor cells), but was also expressed by tumor-infiltrating lymphocytes (TIL; 18 of 44, 41%). Interestingly, intratumor expression of B7-H1 was significantly associated with histologic grade III-negative (P = .012), estrogen receptor-negative (P = .036), and progesterone receptor-negative (P = .040) patients. In addition, the expression of B7-H1 in TIL was associated with large tumor size (P = .042), histologic grade III (P = .015), positivity of Her2/neu status (P = .019), and severe tumor lymphocyte infiltration (P = .001). Taken together, these data suggest that B7-H1 may be an important risk factor in breast cancer patients and may represent a potential immunotherapeutic target using monoclonal antibody against the B7-H1 molecule.