Stereo- and regioselectivity of hepatic oxidation in man — Effect of the debrisoquine/sparteine phenotype on bufuralol hydroxylation

Summary The influence of the debrisoquine/sparteine-type of oxidation polymorphism on plasma bufuralol concentration and the pattern of urine metabolites was studied in extensive and poor metabolizer subjects. (+)- and (–)-bufuralol, and (+)- and (–)-OH-bufuralol in plasma were determined by enantioselective HPLC, and urinary bufuralol and its metabolites were assayed by gas chromatography-mass spectrometry. Three hours after administration of racemic bufuralol the plasma (–)/(+) isomeric ratio for unchanged bufuralol was 1.84 in extensive metabolizers, indicating preferential clearance of the (+)-isomer through aliphatic 1-hydroxylation and glucuroconjugation, while the (–)-isomer was mainly eliminated by aromatic 4-hydroxylation. Poor metabolizers were characterized by impaired 1- and 4-hydroxylation, with almost total abolition of the stereoselectivity of these reactions. The data strongly suggest that both 1- and 4-hydroxylation are catalyzed by the same enzyme. These in vivo observations are in agreement with recent in vitro data obtained in human liver microsomes from phenotyped patients and support the concept of deficiency of a highly stereoselective cytochrome P-450 isozyme as the cause of this polymorphism.     

CYP2D6 gene polymorphism in caucasian smokers: lung cancer susceptibility and phenotype-genotype relationships

Individual susceptibility to smoking-related cancers is proposed to partly depend on a genetically determined ability to metabolise tobacco carcinogens. We previously reported on the association between the activity of the xenobiotic-metabolising enzyme CYP2D6 and lung cancer risk in a hospital-based case-control study among French Caucasian smokers. Here we extended the study to address the effect of four gene-inactivating mutations (CYP2D6(*)3, (*)4, (*)5 and (*)16) and the gene duplication of the CYP2D6 gene (CYP2D6(*)2x2 or CYP2D6(*)1x2) on lung cancer risk in the same population (150 patients with primary lung carcinoma of squamous cell or small cell histology and 172 controls). The risk of lung cancer associated with the CYP2D6 poor metaboliser genotype (odds ratio 1.5, 95% confidence interval 0.5-4.3) did not differ from that in the reference category of extensive metaboliser and ultra-rapid metaboliser genotypes combined. Lung cancer risks for the CYP2D6 PM genotype amongst light smokers (tobacco consumption 20 g/day) were not significantly different. The present findings agree with the discrepancy between the phenotype-based and genotype-based studies indicated by the recent meta-analyses.     

Primary squamous cell carcinoma of the breast: Sensitivity to cisplatinum-based chemotherapy

Primary squamous cell carcinoma (SCC) of the breast is a rare malignancy whose optimal treatment and prognosis anre unknown. A patient with SCC whose tumor responded dramatically to chemotherapy as part of multimodal treatment is presented. A 61-year-old woman had a palpable 5.5-cm tender left breast mass with overlying skin edema and erythema and irregular margins by mammography. Fine needle aspiration revealed malignant squamous cells with keratinization; incisional biopsy confirmed SCC. Extensive evaluation for an extramammary primary site of disease was negative. Neoadjuvant cisplatinum and 5-fluorouracil (5-FU) led to tumor shrinkage and complete resolution of pain and erythema. Modified radical mastectomy with post-operative chest wall radiation were performed. Neither residual invasive carcinoma nor metastatic nodal disease was found, though intraductal carcinoma with marked squamous features was identified. The patient remains disease-free 2.5 years after diagnosis. Cisplatinum-based chemotherapy should be considered in the treatment regimen of this disease.     

Human recombinant interleukin 1 stimulates collagenase and prostaglandin E2 production by human synovial cells.

The pathogenesis and progression of rheumatoid arthritis involves the production of biologically active lymphokines and monokines. Of these, interleukin 1 (IL-1) has been somewhat of a controversial molecule because it seems to evoke various biological responses in several different tissues. In these studies we demonstrate that three biological properties of human monocyte-derived IL-1 (T-lymphocyte activation and human synovial cell prostaglandin E2 and collagenase production) co-purify. The complementary DNA for the prominent pI 7 form of human IL-1 was expressed, purified, and tested. Any controversy now appears resolved since homogeneous recombinant human IL-1 stimulates prostaglandin E2 and collagenase from human synovial cells as well as activates T cells in vitro.     

Tumor necrosis factor and interleukin-1 activities in free lung cells after single and repeated inhalation of bacterial endotoxin.

Bacterial endotoxins (lipopolysaccharides), important components of many organic dusts, are known to induce macrophages to produce the inflammatory mediators interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). To investigate the role of these mediators in the early inflammatory responses in the lung, guinea pigs were exposed to an aerosol of bacterial endotoxin. A bronchoalveolar lavage (BAL) was then performed, and TNF-alpha and IL-1 in lysed BAL cells and in the supernatants from BAL cell cultures were studied. The effect of single and repeated LPS inhalation exposures on the activities of TNF and IL-1 was studied, as was the effect of LPS added to the cell culture medium. A single inhalation exposure to LPS caused an increase in the TNF-alpha and IL-1 activities in cell lysate and in the cell culture supernatant. After a second inhalation exposure, cell-associated and extracellular TNF-alpha activity could not be detected, whereas IL-1 activity was markedly enhanced. IL-1 activity was increased when LPS was added to the cell culture medium with or without a prior inhalation exposure. In contrast, TNF-alpha activity was not affected after a second exposure.     

Induction of human interleukin 1 mRNA measured by collagenase- and prostaglandin E2-stimulating activity in rheumatoid synovial cells

Human blood peripheral monocyte/macrophages release in culture a mononuclear cell factor (MCF) which stimulates the production of collagenase and prostaglandin E2 by human rheumatoid synovial cells and dermal fibroblasts. These two products play a role in connective tissue destruction. MCF has an apparent molecular weight of approximately 15 000 and is biologically and biochemically indistinguishable from interleukin 1. MCF therefore belongs to the well-documented nonimmune biological activities attributed to interleukin 1. Studies on the mechanisms of production and action of such monokine(s) have been difficult in view of the minute quantities produced by freshly isolated cells or from human monocytic lines. Starting from lectin-stimulated human blood mononuclear cells, we have isolated poly(A)+ RNA and studied its translation following microinjection into Xenopus laevis oocytes. The mRNA translation products stimulated collagenase and prostaglandin E2 production in human rheumatoid synovial cells and dermal fibroblasts. The size of MCF-mRNA was estimated to be 10 S. The mRNA of a member of the interleukin 1 family can now be studied in a system based on a specific and direct relevant biological assay and eventually compared with those of other monokines.     

Rheumatoid synovial cell morphologic changes induced by a mononuclear cell factor in culture

Adherent rheumatoid synovial cells in culture produce large amounts of prostaglandin E₂ (PGE₂) and collagenase. When exposed to a monocyte-derived factor, such cells exhibit marked increases in PGE₂ and collagenase production. In addition, cellular morphology becomes more stellate. In the presence of this factor, indomethacin inhibits both PGE₂ production and the stellate changes, whereas collagenase production usually continues at a high rate. Addition of PGE₂ to cultures reproduces the stellate change as does the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP. Colchicine inhibits morphologic transformation induced by the monocyte-derived factor, whereas cytochalasin B has no effect. It appears that the stellate morphology is dependent upon PGE₂-induced cAMP stimulation and is not related to collagenase production per se.     

Axillary sentinel lymph node examination in breast carcinoma.

OBJECTIVE: To evaluate whether the type of pathologic examination of breast sentinel nodes (frozen section, step sections, and immunoperoxidase staining) results in different percentages of nodes positive for metastatic disease. DESIGN: Twenty-eight consecutive patients with breast sentinel node biopsies were evaluated by step-sectioning the sentinel node(s) along with performing immunoperoxidase stains for low-molecular-weight cytokeratin and epithelial membrane antigen. SETTING AND PARTICIPANTS: The patients were from a university hospital and large private hospital. MAIN OUTCOME MEASURES: The results of the step sections and immunoperoxidase stains were compared with routine examination, that is, intraoperative frozen section along with a single hematoxylin-eosin slide. RESULTS: Nine cases were positive by routine evaluation, 10 by step sections, and 11 by immunoperoxidase staining. CONCLUSIONS: The large, multi-institutional studies of sentinel node utility must take into account the surgical pathology methods used to evaluate these specimens so that uniform techniques, which reliably predict the status of the axillary nodes, can be instituted at all institutions that use this procedure.     

Grapevines under drought do not express esca leaf symptoms

In the context of climate change, plant mortality is increasing worldwide in both natural and agroecosystems. However, our understanding of the underlying causes is limited by the complex interactions between abiotic and biotic factors and the technical challenges that limit investigations of these interactions. Here, we studied the interaction between two main drivers of mortality, drought and vascular disease (esca), in one of the world’s most economically valuable fruit crops, grapevine. We found that drought totally inhibited esca leaf symptom expression. We disentangled the plant physiological response to the two stresses by quantifying whole-plant water relations (i.e., water potential and stomatal conductance) and carbon balance (i.e., CO₂ assimilation, chlorophyll, and nonstructural carbohydrates). Our results highlight the distinct physiology behind these two stress responses, indicating that esca (and subsequent stomatal conductance decline) does not result from decreases in water potential and generates different gas exchange and nonstructural carbohydrate seasonal dynamics compared to drought.

For many plant pathogens, it is still largely unknown if their interactions with abiotic stresses are synergistic, antagonistic, or neutral. These interactions are particularly crucial in the case of vascular diseases and drought (1–3). Both affect the same plant tissue, the xylem vascular network, which is responsible for the movement of water and nutrients throughout the plant. A strong synergy when combining drought and vascular disease could accelerate plant death (1, 3) and has strong implications in the context of climate change, in which a global increase of drought and associated plant mortality is expected (4).Grapevine, one of the most economically valuable crops in the world (5), is being threatened by future climate change scenarios (6). Since the early 2000s, old-world vineyards have exhibited increasing yield losses, and although the causes are not completely understood, an increased incidence of trunk diseases has been identified as one of the main contributors (7, 8). One of the most prominent of these diseases is esca. Esca is a vascular disease associated with losses in fruit quality and quantity and increased vine mortality, and the mechanisms of esca pathogenesis are still largely misunderstood (9, 10). This latent disease primarily affects perennial organs (i.e., the trunk), causing necrosis of internal tissues. Annual organs (i.e., leaves and clusters) typically begin to display symptoms in mature plants (normally older than 10 y) (11, 12). Recent work by our laboratory quantified the presence of hydraulic failure in the xylem tissue of esca-symptomatic leaves and stems (13, 14), and it is hypothesized that the transpiration stream facilitates the transport of phytotoxic metabolites from the pathogen niche in the trunk to the leaves (8). Therefore, the physiology controlling vine water use likely plays a crucial role during esca pathogenesis. In this context, we hypothesized a strong interaction between esca and drought, which both affect xylem water transport. Drought events cause yield decline, and when severe and/or prolonged, vine mortality (15, 16). Due to their climatic and edaphic environment, most of the world’s wine regions are exposed to a high risk of drought, as irrigation is not a sustainable long-term solution and rainfall is often not sufficient to supply grapevine evapotranspiration [e.g., in the Mediterranean area (17, 18)]. Because both drought and esca are associated with xylem hydraulic failure (13, 14, 19) and, theoretically, with nonstructural carbohydrate (NSC) consumption (1, 3, 8), these stresses could synergize and amplify the current vineyard decline. Therefore, there are real and urgent concerns regarding the outcome of the interaction between drought events and vascular pathogenesis in the grapevine.On some levels, plant responses to vascular disease and drought appear similar and include decreases in leaf gas exchange (20–22), losses of hydraulic conductivity (14, 23, 24), wilting (i.e., decreases in cell turgor), and scorching of leaves (12, 25, 26). Because they induce similar plant responses, it could be assumed that their interactions would be synergistic. However, we lack the detailed whole-plant physiology studies necessary to determine if this is true. This is probably because studying disease–drought interaction can be extremely challenging. Some vascular diseases, including esca, cannot be reproduced through the artificial inoculation of plants. Thus, studies rely on naturally infected field experimentation in which the disease history of specific plants is largely unknown and applying well-controlled water deficits (WD) (as well as controlling other environmental factors) is either difficult or impossible.The main objective of this study was to explore the interaction between esca and drought and to disentangle the whole-plant physiological response to the two stresses. We overcame the technical barriers by transplanting naturally infected plants with known disease histories from the field into pots to precisely manipulate their watering regime and study the combined effect of esca and drought. We maintained half of the plants under a WD at a predawn water potential (Ψ_PD) ∼ −1 MPa during 3 mo in two consecutive seasons, which simulated a moderate to severe level of drought (27). During these periods, we quantified plant–water relations (water potential and whole-plant and leaf-stomatal conductance), carbon balance (CO₂ assimilation, chlorophyll content, and NSC quantification in leaves and stems), and the development of esca symptoms. Our results showed that whole-plant physiological responses to esca or drought are not driven by the same underlying mechanisms; once combined, the two stresses strongly and antagonistically interact, opening perspectives on the plant–pathogen–environment relationships impacting vineyard sustainability.