H+/K+ATPase Expression in the Larynx of Laryngopharyngeal Reflux and Laryngeal Cancer Patients.

OBJECTIVES: The gastric H+/K+ ATPase proton pump has previously been shown to be expressed in the human larynx, however its contribution to laryngopharyngeal reflux (LPR) signs, symptoms and associated diseases such as laryngeal cancer is unknown. Proton pump expression in the larynx of patients with LPR and laryngeal cancer was investigated herein. A human hypopharyngeal cell line expressing the proton pump was generated to investigate its effects. STUDY DESIGN: In-vitro translational. METHODS: Laryngeal biopsies were obtained from three LPR and eight LSCC patients. ATP4A, ATP4B and HRPT1 were assayed via qPCR. Human hypopharyngeal FaDu cell lines stably expressing proton pump were created using lentiviral transduction and examined via transmission electron microscopy and qPCR for genes associated with inflammation or laryngeal cancer. RESULTS: Expression of ATP4A and ATP4B was detected in 3/3 LPR, 4/8 LSCC-tumor and 3/8 LSCC-adjacent specimens. Expression of ATP4A and ATP4B in FaDu elicited mitochondrial damage and expression of IL1B, PTGS2, and TNFA (P < .0001); expression of ATP4B alone did not. CONCLUSIONS: Gastric proton pump subunits are expressed in the larynx of LPR and LSCC patients. Mitochondrial damage and changes in gene expression observed in cells expressing the full proton pump, absent in those expressing a single subunit, suggest that acid secretion by functional proton pumps expressed in upper airway mucosa may elicit local cell and molecular changes associated with inflammation and cancer. LEVEL OF EVIDENCE: NA Laryngoscope, 131:130-135, 2021.

Presence of pepsin in laryngeal tissue and saliva in benign and malignant neoplasms.

OBJECTIVES: The current study was performed to determine the presence of pepsin in saliva and laryngeal tissue among participants with benign and malignant laryngeal neoplasms. STUDY DESIGN: Case-control study included three groups of patients with: (1) benign laryngeal neoplasms, (2) malignant laryngeal neoplasms and (3) control subjects without symptoms or signs of laryngopharyngeal reflux (LPR). METHODS: Eighty-one voluntary participants were included into study. They were recruited from a group of patients with histologically proven benign and malignant laryngeal neoplasms and in case of control subjects among patients with nasal septum deformation without symptoms of LPR. Morning saliva samples were collected preoperatively. Tumor biopsies were collected by directoscopy of larynx and the control samples from interarytenoid unit of larynx. All samples were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) and Immunohistochemistry. RESULTS: Pepsin was found in all samples of saliva and tissue biopsies in groups with malignant and benign neoplasms. The highest concentration of pepsin was found in a group of patients with malignant laryngeal neoplasms. Patients with benign laryngeal neoplasms had lower concentrations and the control subjects presented with the lowest concentration of pepsin measured from their saliva. Differences were not statistically significant. Immunohistochemical (IHC) analysis showed the largest number of high positive samples in the group of malignant lesions. CONCLUSION: These results suggest that pepsin and LPR can contribute to the development of benign and malignant laryngeal neoplasms. Further prospective studies, with far more patients, are necessary to prove the role of pepsin in multifactorial etiology of laryngeal neoplasms.

Expression of nNOS in the human larynx.

Although intrinsic laryngeal neurons and ganglia have been studied in various species, they have been overlooked in humans. We aimed to investigate the presence of intrinsic laryngeal neurons in humans and, if present, to analyze their neuronal nitric oxide synthase (nNOS) expression. An immunohistochemical study using anti-nNOS antibodies was performed on samples obtained from four cadavers. Intrinsic laryngeal nNOS+ neurons were assessed in the submucosal layer, but nNOS+ nerves were found in all histological layers of the larynx. nNOS expression was also found in striated muscle fibers of larynx. This might reveal the anatomical basis of an upwards extension of the nonadrenergic noncholinergic system in human airways, but further experiments are needed to assess an exact role of NO influence on neural transmission and muscular functions of human larynx.

New aspects in the pathomechanism and diagnosis of the laryngopharyngeal reflux-clinical impact of laryngeal proton pumps and pharyngeal pH metry in extraesophageal gastroesophageal reflux disease.

AIM: To determine the laryngeal H+K+-ATPase and pharyngeal pH in patients with laryngopharyngeal reflux (LPR)-symptoms as well as to assess the symptom scores during PPI therapy. METHODS: Endoscopy was performed to exclude neoplasia and to collect biopsies from the posterior cricoid area (immunohistochemistry and PCR analysis). Immunohistochemical staining was performed with monoclonal mouse antibodies against human H+K+-ATPase. Quantitative real-time RT-PCR for each of the H+K+-ATPase subunits was performed. The pH values were assessed in the aerosolized environment of the oropharynx (DxpH Catheter) and compared to a subsequently applied combined pH/MII measurement. RESULTS: Twenty patients with LPR symptoms were included. In only one patient, the laryngeal H+K+-ATPase was verified by immunohistochemical staining. In another patient, real-time RT-PCR for each H+K+-ATPase subunit was positive. Fourteen out of twenty patients had pathological results in DxpH, and 6/20 patients had pathological results in pH/MII. Four patients had pathological results in both functional tests. Nine out of twenty patients responded to PPIs. CONCLUSION: The laryngeal H+K+-ATPase can only be sporadically detected in patients with LPR symptoms and is unlikely to cause the LPR symptoms. Alternative hypotheses for the pathomechanism are needed. The role of pharyngeal pH-metry remains unclear and its use can only be recommended for patients in a research study setting.

[Expression and significance of DNA-dependent protein kinase in human laryngeal squamous cell carcinoma].

OBJECTIVE: To study the expression of DNA-dependent protein kinase (DNA-PK) in human laryngeal squamous cell carcinoma (LSCC) and normal laryngeal mucosa (NLM), and to analysize the relationship between the expression and the clinicopathologic parameters of LSCC. METHOD: Immunohistochemical technique (Envision) was used to detect the expression of DNA-PK in 64 cases of LSCC and 15 cases of NLM. To investigate an investigation was conducted on the relationship between the expression and clinico-pathological features of LSCC. RESULT: DNA-PK was lowly expressed in NLM and highly expressed in LSCC,the positive rate of DNA-PK expression was 26.67% (4/15), 78.13% (50/64), respectively, and there was significant different difference between the two groups (P < 0.05). Its expression was correlated with the level of histodifferentiation (P < 0.05), but not with TNM stages and neck lymph node metastasis (P > 0.05). CONCLUSION: DNA-PK may be involved in disease development of LSCC.

Expressions of Src homology 2 domain-containing phosphatase and its clinical significance in laryngeal carcinoma.

We investigated the expression of Src homology 2 domain-containing phosphatase (SHP-2) in laryngeal carcinoma and its clinical significance. Expression of SHP-2 was detected by immunohistochemical staining in normal mucosal tissues and various grades of laryngeal carcinoma. We looked for possible correlations between expression of SHP-2 in laryngeal carcinoma and clinical staging and lymph node metastasis. Immunochemical staining results revealed that the SHP-2 expression was significantly higher (88.24%) in laryngeal carcinoma than in normal mucosal tissue (25%). Additionally, the expression of SHP-2 was significantly correlated with lymph node metastasis, but not with clinical stage and gender of patients with laryngeal carcinoma. Therefore, SHP-2 may be useful as a prognostic marker for laryngeal carcinoma and as a therapeutic target in laryngeal carcinoma treatment.

Expression of matrix metalloproteinase-9 in patients with squamous cell carcinoma of the larynx.

Aim of this study was to investigate the correlation of matrix metalloproteinase-9 (MMP-9) expression with histopathologic and clinical characteristics of laryngeal squamous cell carcinoma, and to assess the role of MMP-9 expression in patient survival. Study included 196 patients with squamous cell carcinoma of the larynx treated at ENT Department, Split University Hospital Centre, from January 1, 2000 till December 31, 2009. The level of MMP-9 expression showed a statistically significant correlation (p < 0.001) with the disease histopathologic grade, stage, metastatic potential, recurrence potential, and survival. Kaplan-Meier curve yielded a statistically significant survival difference (p < 0.001) among the three patient groups with different levels of MMP-9 expression. The survival curve clearly showed the MMP-9 expression as a predictor of survival to be significantly (p < 0.001) associated with survival. In this study, MMP-9 expression as a biological marker showed a potential predictive value in laryngeal squamous cell carcinoma.

Expression and distribution of aggrecanases in human larynx: ADAMTS-5/aggrecanase-2 is the main aggrecanase in laryngeal carcinoma.

Members of the ADAMTS family of proteases degrade proteoglycans and thereby have the potential to alter tissue architecture and regulate cellular functions. Aggrecanases are the main enzymes responsible for aggrecan degradation, due to their specific cleavage pattern. In this study, the expression status, the macromolecular organization and localization of ADAMTS-1, ADAMTS-4/aggrecanase-1 and ADAMTS-5/aggrecanase-2 in human normal larynx and laryngeal squamous cell carcinoma (LSCC) were investigated. On mRNA level, the results showed that ADAMTS-4 was the highest expressed enzyme in normal larynx, whereas ADAMTS-5 was the main aggrecanase in LSCC presenting a stage-related increase up to stage III (8-fold higher expression compared to normal), and thereafter decreased in stage IV. Accordingly, immunohistochemical analysis showed that ADAMTS-5, but not ADAMTS-4, was highly expressed by carcinoma cells. Sequential extraction revealed an altered distribution and organization of multiple molecular forms (latent, activated and fragmented forms) of the enzymes within the cancerous and their corresponding macroscopically normal laryngeal tissues, compared to the normal ones. Importantly, these analyses indicated that critical macromolecular changes occurred from the earliest LSCC stages not only in malignant parts of the tissue but also in areas that were not in proximity to carcinoma cells and appeared otherwise normal. Overall, the results of the present study show that ADAMTS-5/aggrecanase-2 is the main aggrecanase present in laryngeal carcinoma suggesting a critical role for the enzyme in aggrecan degradation and laryngeal tissue destruction during tumor progression.

Cyclooxygenase-2 expression and clinical parameters in laryngeal squamous cell carcinoma, vocal fold nodule, and laryngeal atypical hyperplasia.

BACKGROUND: The diagnostic role of cyclooxygenase-2 (COX-2) expression in laryngeal atypical hyperplasia, vocal fold nodule, and laryngeal squamous cell carcinoma was examined. METHODS: Specimens obtained from patients diagnosed with vocal fold nodule (n = 35), atypical hyperplasia (n = 35), laryngeal squamous cell carcinoma (n = 35), and clinical parameters were evaluated retrospectively. RESULTS: Although no staining was observed in patients with vocal fold nodules, staining was noted in laryngeal atypical hyperplasia and squamous cell carcinoma. The percentage of COX-2 staining was the highest in the carcinoma group. CONCLUSION: It was determined that COX-2 staining was significantly associated with laryngeal squamous cell carcinoma. It should be noted that overexpression of COX-2, a potentially important factor in the evolution of carcinogenesis in precancerous lesions, might be an indicator of the development of carcinoma.

Methionine-centered redox cycle in organs of the aero-digestive tract of young and old rats.

It is commonly accepted that aging is associated with a decline in the antioxidant defense of the cell; accordingly, certain redox enzymes are used as markers of biological senescence. To further test and specify this general concept, we studied age-related changes in the enzymes of the methionine-centered redox cycle (MCRC) in four aero-digestive organs of rats. The levels of cytosolic thioredoxin (Trx), thioredoxin reductase (TrxR), and methionine sulfoxide reductase (Msr), all tended to decline with age. The enzymatic activities of MsrA and MsrB were significantly lower in the organs of aged animals. In general, the magnitude of this decline increased in the order: tongue < sternohyoid muscle < larynx < esophagus. The relative stability of MCRC in the old tongues might be part of the well-preserved oxidative metabolism as confirmed by the age-related increase in mitochondrial marker and muscle tissue in these tongues. In total, the results suggest that age-associated oxidative damage is organ-specific and could reflect differences in morphological composition of these tissues, and among them, relative content of striated muscles.

Systematic expression profiling of the gastric H+/K+ ATPase in human tissue.

OBJECTIVE: The potassium-competitive acid blockers (P-CABs), comprise a new, innovative group of competitive and reversible inhibitors of the gastric H+/K+ ATPase. Our aim was to identify sites of expression of the H+/K+ ATPase that are potential targets of these compounds by examining the expression profile of the gastric H+/K+ ATPase in the human body from a broad range of tissues. MATERIAL AND METHODS: Expression profiling was done by quantitative mRNA analysis (TaqMan PCR). Tissues that were mRNA-positive for the alpha subunit were investigated further by Western blot and immunohistochemistry (IHC) for the presence of gastric H+/K+ ATPase protein. RESULTS: In addition to the very high expression levels in the stomach, the adrenal gland, cerebellum and pancreas gave unexpectedly positive mRNA signals for the alpha subunit of gastric H +/K+ ATPase. However, they were negative for mRNA of the beta subunit, and Western blot and IHC were negative for alpha and beta subunit protein. Another group of tissues with low alpha subunit mRNA expression including the frontal cortex, cortex grey matter, testis, thymus and larynx submucosa were also found negative for both alpha and beta subunit protein. In contrast to mouse kidney, no gastric H+/K+ ATPase could be detected in human kidney. CONCLUSIONS: We therefore conclude that the only organ in humans expressing significant levels of the P-CAB target gastric H+/K+ ATPase is the stomach.

Comparative cytochrome P450 -1A1, -2A6, -2B6, -2C, -2D6, -2E1, -3A5 and -4B1 expressions in human larynx tissue analysed at mRNA level.

The metabolic activation of numerous exogenous and endogenous chemicals is catalysed by cytochrome P450 enzymes (CYPs). The aim of this study was to analyse the expression of the individual forms of CYP at the mRNA level in human larynx and quantitatively to compare their expressions in human liver, the main organ of CYP expression. Individual forms of CYP mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for the CYPs -1A1, -1A2, -2A6, -2B6, -2C, -2D6, -2E1, -3A3/4, -3A5, -3A7 and -4B1. An RNA competitor of known copy number, covering the primer sequences necessary to amplify the entire object CYPs within a single molecule, was used as reference. This study reports a consistent detection of mRNAs for the CYPs -1A1, -2A6, -2B6, -2C, -2D6, -2E1, -3A5 and -4B1 in the human larynx tissue. The data indicate that the human larynx highly resembles the lung tissue in CYP content, as a comparable subset of CYP mRNAs was detected in the larynx previously reported for human lung with the exception of CYP1A2. The results are discussed in quantitative ratios of the detected CYP mRNAs in relation to the hepatic CYP expression.

Selective reduction of extracellular signal-regulated protein kinase (ERK) phosphorylation in squamous cell carcinoma of the larynx.

Mitogen-activated protein kinase (MAPK) cascades transmit and amplify signals involved in cell proliferation as well as in cell death. In this study, the potential derangement of MAPK pathways has been evaluated in human squamous cell carcinomas (SCC) of the larynx. The expression and activity of the MAPK p38, ERK1/2p44/p42 and JNK/SAPKp46/p54 have been investigated by immunoblot analysis of tissue homogenates in 27 samples of primary laryngeal cancer and in 27 paired non-neoplastic laryngeal mucosa. On the same tissues, the activation of MAPK JNK/SAPKp46/p54 was also analyzed by an ELISA assay. The results obtained showed that both total and phosphorylated levels of JNK/SAPKp46/p54 and p38 were not different between tumor and normal samples. Conversely, while total protein levels for both ERK1p44 and ERK2p42 were not statistically different between tumor and normal samples, the analysis of the level of the activated forms of ERK1/2 showed a statistically significant decreased phosphorylation of both isoforms in the tumor samples compared to the control tissues. The rate of reduction was similar for both isoforms. Immunohistochemical analysis of all the activated MAPK (p38, JNK/SAPKp46/p54 and ERK1/2p44/p42) in both laryngeal SCC and normal mucosa demonstrated no difference of cellular localization. Activated ERK1/2p44/p42 and activated p38 demonstrated a nucleo-cytoplasmic distribution whereas activated JNK/SAPKp46/p54 were localized into the cytoplasmic membrane. The decreased activity of ERK1/2p44/42 in laryngeal SCC might reflect alterations in tumor suppressing activity or might derive from the interplay among various transduction pathways.

Heparanase localization and expression by head and neck cancer: correlation with tumor progression and patient survival.

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate (HS) side chains of HS proteoglycans, the major proteoglycans in the extracellular matrix and cell surfaces. Traditionally, heparanase activity was implicated in cellular invasion associated with angiogenesis, inflammation, and cancer metastasis. More recently, heparanase upregulation was documented in an increasing number of primary human tumors, correlating with reduced postoperative survival rate and enhanced tumor angiogenesis. In the present study, we examined the expression of heparanase in squamous cell carcinoma of the head and neck by means of immunostaining, and we correlated expression levels with patient outcome. The intensity and extent of heparanase staining correlated with tumor stage (P = .049 and P = .027, respectively), and the extent of staining further correlated with tumor grade (P = .047). Moreover, heparanase expression inversely correlated with patient status at the end of the study (P = .012). Notably, heparanase localization was found to be an important parameter for patient status. Thus, 63% of patients with nuclear staining, compared to 19% of patients with cytoplasmic staining (P = .0043), were alive, indicating that nuclear localization of the enzyme predicts a favorable outcome.

[Activity of cysteine proteinases and their inhibitors in larynx cancerogenic tumours].

Data on the study of activity of cysteine proteinases and their inhibitors in larynx neoplastic tissues and control mucosa surrounding them are submitted. It is established that cysteine activity was significantly increased and the content of cysteine inhibitors was decreased in the larynx cancer tissue compared to papilloma tissue and control mucosa. The ratios between cysteine activity and level inhibitors is the informative index of the trual potential proteolytic activity in cancer cells and their invasive capacity.

Phosphatidylinositol 3-kinase regulates early differentiation in human laryngeal keratinocytes.

Epidermal growth factor receptor (EGFR) signaling regulates a variety of cellular functions, including proliferation, gene expression, and differentiation. Infection of laryngeal epithelial cells by human papillomaviruses causes recurrent respiratory papillomas, benign tumors characterized by an altered pattern of differentiation. Papilloma cells overexpress the EGFR and have constitutively active extracellular signal-regulated kinase (ERK) and enhanced phosphatidylinositol 3-kinase (PI3K) activity, but overexpression of the lipid phosphatase PTEN (Phosphatase and Tensin Homolog) reduces activation of Akt by PI3K. We hypothesized that the altered differentiation of papillomas reflects these changes in signaling from the EGFR-ERK and PI3K-Akt pathways and that one or both of these pathways is required for the normal differentiation process in mucosal epithelium. Inhibiting either the enzymatic activity or the synthesis of PI3K in uninfected laryngeal cells blocked expression of keratin-13 (K13), a protein induced during normal differentiation. In contrast, inhibiting activation of ERK had minimal effect. Using ribonucleic acid interference to reduce protein levels of integrin-linked kinase 1 or phosphoinositide-dependent protein kinase 1, intermediates in the activation of Akt by PI3K, or reducing levels of Akt-1 itself did not inhibit K13 expression by normal laryngeal keratinocytes. We conclude that PI3K activation is an important regulator of expression of K13, a marker for the normal differentiation process in human mucosal keratinocytes, that this function does not require activation of Akt-1, and that the failure to express K13 in papilloma cells is not because of reduction in activated Akt.

Telomerase catalytic subunit in laryngeal carcinogenesis--an immunohistochemical study.

We recently demonstrated that (1) telomerase catalytic subunit messenger RNA (mRNA) relative quantities increase progressively with the degree of laryngeal epithelial abnormalities and that (2) telomerase catalytic subunit gene re-expression represents an early event in laryngeal carcinogenesis. The aim of the study was to determine whether telomerase catalytic protein immunohistochemisty reflects telomerase catalytic subunit gene expression in different grades of laryngeal epithelial abnormalities and squamous cell carcinomas of the larynx. Telomerase catalytic protein was analysed immunohistochemically in 106 laryngeal epithelial tissue samples: 10 normal epithelia, 15 squamous cell hyperplasias, 14 basal/parabasal cell hyperplasias, 10 atypical hyperplasias, eight intraepithelial carcinomas and 49 squamous cell carcinomas. At least 200 nuclei of each lesion were quantified per slide and the number of positive signals per nucleus was expressed as a telomerase catalytic protein index. The mean telomerase catalytic protein index increased progressively with the degree of laryngeal epithelial abnormalities: from 0.17 in normal epithelia, 0.44 in squamous cell hyperplasia, 0.54 in basal/parabasal cell hyperplasia, 0.91 in atypical hyperplasia, 1.05 in intraepithelial carcinoma to 0.96 in squamous cell carcinomas. Statistical analysis revealed three different groups of laryngeal epithelial changes according to the number of telomerase catalytic protein signals per nucleus: (1) normal epithelium, (2) regenerative epithelium (squamous cell hyperplasia, basal/parabasal cell hyperplasia), and (3) atypical hyperplasia, intraepithelial carcinoma and squamous cell carcinoma (P<0.0033). Telomerase catalytic protein immunohistochemistry parallels well with telomerase catalytic subunit mRNA relative quantities in laryngeal carcinogenesis. In normal and regenerative laryngeal epithelia, telomerase catalytic protein is present in occasional basal/parabasal nuclei, becomes undetectable with maturation or differentiation of epithelial cells, and reflects the regenerative capacity of squamous epithelium. Nevertheless, several telomerase catalytic protein signals in the majority of nuclei in precancerous lesions, intraepithelial carcinomas and squamous cell carcinomas, are consistent with telomerase catalytic subunit gene re-expression, an early event in laryngeal carcinogenesis.

[Hypermethylation of the death-associated protein kinase promoter in laryngeal squamous cell cancer].

OBJECTIVE: To explore the relationship between hypermethylation of the promoter of death-associated protein kinase (DAPK) gene and laryngeal squamous cell cancer. METHODS: Promoter hypermethylation and mRNA expression of DAPK gene were detected by methylation-specific PCR, RT-PCR and gene sequencing. RESULTS: Among the 58 patients with laryngeal squamous cell cancer, hypermethylation of DAPK promoter was detected in 39 cases (67.2%). There was no significant difference in hypermethylation in relation to pathological grade and clinical staging, but a highly significant difference was observed between patients with and without lymph node metastasis (N0 and N1) (P < 0.001). DAPK promoter hypermethylation was detected in tumor adjacent tissues in 6 of the 58 cases. DAPK mRNA was not expressed in all laryngeal squamous cell cancers having hypermethylation of DAPK promoter, whereas it was expressed in normal laryngeal mucosa, laryngeal squamous cell cancers without hypermethylation and tumor adjacent tissues. CONCLUSION: Hypermethylation of DAPK promoter is associated with loss of its transcription in laryngeal squamous cell carcinoma. The high frequency hypermethylation of DAPK promoter illustrates its potential clinical application as tumor marker for diagnosis and prognosis.

Characterization of benzo(a)pyrene-trans-7,8-dihydrodiol glucuronidation by human tissue microsomes and overexpressed UDP-glucuronosyltransferase enzymes.

UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of benzo(a)pyrene-trans-7,8-dihydrodiol (BPD), precursor to the potent mutagen benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, may be an important pathway in the detoxification of benzo(a)pyrene. To better characterize this pathway in humans, high-pressure liquid chromatography (HPLC) was used to detect glucuronide conjugates of BPD formed in vitro. Three peaks were detected by HPLC after incubation of racemic BPD with human liver microsomes; these were identified as monoglucuronides by liquid chromatography-mass spectrometry analysis. Proton nuclear magnetic resonance spectroscopy of isolated fractions, combined with HPLC analysis of the glucuronide products from human liver microsomal incubations with purified benzo(a)pyrene-trans-7S,8S-dihydrodiol [(+)-BPD] and benzo(a)pyrene-trans-7R,8R-dihydrodiol [(-)-BPD] forms of BPD, indicated that peak 1 contained the 7-glucuronide of 7S,8S-BPD (BPD-7S-Gluc), peak 2 was a mixture of the 7-glucuronide of 7R,8R-BPD (BPD-7R-Gluc) and the 8-glucuronide of 7S,8S-BPD (BPD-8S-Gluc), and peak 3 contained the 8-glucuronide of 7R, 8R-BPD (BPD-8R-Gluc). In liver microsomes, peak 1 (BPD-7S-Gluc) was the largest peak observed, whereas in microsomes from aerodigestive tract tissues, peak 2 (both BPD-7R-Gluc and BPD-8S-Gluc) was the largest HPLC peak observed. The liver enzymes UGT1A1 and UGT2B7 formed BPD-7S-Gluc as the major diastereomer, whereas UGT1A8 and UGT1A10, extrahepatic enzymes present in the aerodigestive tract, preferentially formed both BPD-7R-Gluc and BPD-8S-Gluc. In addition, both UGT1A9 and UGT1A7 preferentially formed BPD-7R-Gluc. No detectable glucuronidating activity against BPD was observed by UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B15, or UGT2B17. The affinity of individual UGT enzymes as determined by K(m) analysis was UGT1A10 > UGT1A9 > UGT1A1 > UGT1A7 for (-)-BPD and UGT1A10 > UGT1A9 > UGT2B7 approximately UGT1A1 > UGT1A7 for (+)-BPD. These results suggest that several UGTs may play an important role in the overall glucuronidation of BPD in humans, with UGT1A1, UGT1A7, UGT1A9, UGT1A10 and potentially UGT1A8 playing an important role in the glucuronidation of the procarcinogenic (-)-BPD enantiomer, and that the stereospecific activity exhibited by different UGTs against BPD is consistent with tissue-specific patterns of BPD glucuronide diastereomer formation and UGT expression.