Robert Langenbach

Opposite effects of cyclooxygenase-1 and -2 activity on the pressor response to angiotensin II

Therapeutic use of cyclooxygenase-inhibiting (COX-inhibiting) nonsteroidal antiinflammatory drugs (NSAIDs) is often complicated by renal side effects including hypertension and edema. The present studies were undertaken to elucidate the roles of COX1 and COX2 in regulating blood pressure and renal function. COX2 inhibitors or gene knockout dramatically augment the pressor effect of angiotensin II (Ang II). Unexpectedly, after a brief increase, the pressor effect of Ang II was abolished by COX1 deficiency (either inhibitor or knockout). Ang II infusion also reduced medullary blood flow in COX2-deficient but not in control or COX1-deficient animals, suggesting synthesis of COX2-dependent vasodilators in the renal medulla. Consistent with this, Ang II failed to stimulate renal medullary prostaglandin E(2) and prostaglandin I(2) production in COX2-deficient animals. Ang II infusion normally promotes natriuresis and diuresis, but COX2 deficiency blocked this effect. Thus, COX1 and COX2 exert opposite effects on systemic blood pressure and renal function. COX2 inhibitors reduce renal medullary blood flow, decrease urine flow, and enhance the pressor effect of Ang II. In contrast, the pressor effect of Ang II is blunted by COX1 inhibition. These results suggest that, rather than having similar cardiovascular effects, the activities of COX1 and COX2 are functionally antagonistic.

Allergic lung responses are increased in prostaglandin H synthase–deficient mice

To investigate the function of prostaglandin H synthase-1 and synthase-2 (PGHS-1 and PGHS-2) in the normal lung and in allergic lung responses, we examined allergen-induced pulmonary inflammation and airway hyperresponsiveness in wild-type mice and in PGHS-1(-/-) and PGHS-2(-/-) mice. Among nonimmunized saline-exposed groups, we found no significant differences in lung function or histopathology, although PGE(2) was dramatically reduced in bronchoalveolar lavage (BAL) fluid from PGHS-1(-/-) mice, relative to wild-type or PGHS-2(-/-) mice. After ovalbumin sensitization and challenge, lung inflammatory indices (BAL cells, proteins, IgE, lung histopathology) were significantly greater in PGHS-1(-/-) mice compared with PGHS-2(-/-) mice, and both were far greater than in wild-type mice, as illustrated by the ratio of eosinophils in BAL fluid (8:5:1, respectively). Both allergic PGHS-1(-/-) and PGHS-2(-/-) mice exhibited decreased baseline respiratory system compliance, whereas only allergic PGHS-1(-/-) mice showed increased baseline resistance and responsiveness to methacholine. Ovalbumin exposure caused a modest increase in lung PGHS-2 protein and a corresponding increase in BAL fluid PGE(2) in wild-type mice. We conclude that (a) PGHS-1 is the predominant enzyme that biosynthesizes PGE(2) in the normal mouse lung; (b) PGHS-1 and PGHS-2 products limit allergic lung inflammation and IgE secretion and promote normal lung function; and (c) airway inflammation can be dissociated from the development of airway hyperresponsiveness in PGHS-2(-/-) mice.

Distinct roles of prostaglandin H synthases 1 and 2 in T-cell development

Prostaglandin G and H synthases, or cyclooxygenases (COXs), catalyze the formation of prostaglandins (PGs). Whereas COX-1 is diffusely expressed in lymphoid cells in embryonic day 15.5 thymus, COX-2 expression is sparse, apparently limited to stromal cells. By contrast, COX-2 is predominant in a subset of medullary stromal cells in three- to five-week-old mice. The isozymes also differ in their contributions to lymphocyte development. Thus, experiments with selective COX-1 inhibitors in thymic lobes from normal and recombinase-activating gene-1 knockout mice support a role for this isoform in the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP). Concordant data were obtained in COX-1 knockouts. Pharmacological inhibition and genetic deletion of COX-2, by contrast, support its role during early thymocyte proliferation and differentiation and, later, during maturation of the CD4 helper T-cell lineage. PGE2, but not other PGs, can rescue the effects of inhibition of either isoform, although it acts through distinct EP receptor subtypes. COX-dependent PG generation may represent a mechanism of thymic stromal support for T-cell development.

Human cell lines, derived from AHH-1 TK+/- human lymphoblasts, genetically engineered for expression of cytochromes P450

We are developing a panel of human B lymphoblastoid cells which have been engineered to express specific human cDNAs for cytochrome P450 and other xenobiotic metabolizing enzymes. The recipient cells are of a human B lymphoblastoid cell line, designated AHH-1 TK+/-. These cells are transfected using two extrachromosomal vectors both containing OriP sequences derived from Epstein Barr virus but containing independent means of selection in mammalian cells. Using this system, the level of cDNA expression is nearly always stable and consistent from one transfection to another. Thus, once the level of expression has been characterized, cell lines with potentially interesting combinations of xenobiotic-metabolizing enzymes can be predictably developed. cDNAs encoding the following human enzymes have been expressed in this system: CYP1A1, CYP1A2, CYP2A6, CYP2B8, CYP2C6, CYP2C9, CYP2D6, CYP2E1, CYP3A4 and microsomal epoxide hydrolase. We have expressed all of these enzymes individually and have developed cell lines which express combinations of the xenobiotic metabolizing enzymes. The expression of multiple enzymes is important for generalized use of engineered cells as toxicology screening tools. We have primarily used the cell lines in applications to toxicology focusing on procarcinogen activation as detected in assays for the induction of gene locus mutations. In this chapter we discuss the general properties of the system and applications to toxicology testing.

Recombinant DNA approaches for the development of metabolic systems used in in vitro toxicology

In the past few years there has been considerable progress in the development of mammalian cell systems for use in genetic toxicology by the stable transfer of genes/cDNAs coding for drug metabolizing enzymes directly into the target cell. Alternative approaches have also been developed in which mammalian cells are transiently transfected with cDNAs coding for drug-metabolizing enzymes and S9 preparations expressing a single metabolizing enzyme isolated and used for metabolic activation. Progress in these areas is reviewed here and the relative merits of the different approaches are discussed. Work to date has focused primarily on the cytochrome P450 family of enzymes, although other enzyme systems involved in xenobiotic metabolism have been used. The central theme of this review is the transfer of genetic information to improve the metabolic capability of cell systems used in genetic toxicology. However, a basic philosophy of the review is that genetic manipulation of cultured mammalian cells has the potential for developing systems to be used to better understand chemically induced toxicological effects.

Cyclooxygenase-2 deficiency increases epidermal apoptosis and impairs recovery following acute UVB exposure.

The cyclooxygenases, COX‐1 and COX‐2, are involved in cutaneous responses to both acute and chronic UV exposure. In the present study, wild‐type (WT), COX‐1−/− and COX‐2−/− mice were used to determine the influence of the individual isoform on mouse skin responses to acute UVB treatment. Immunohistochemistry and Western analysis indicated that COX‐2, and not COX‐1, was induced by UVB (2.5 or 5.0 kJ/m2), but that COX‐1 remained the major source of prostaglandin E2 production. UVB exposure significantly increased epidermal apoptosis in all genotypes compared to untreated mice. However, while the number of apoptotic cells in WT and COX‐1−/− mice were about equal, the number of apoptotic cells was 2.5‐fold greater in COX‐2−/− mice. Apoptosis in WT and COX‐2−/− mice peaked at 24 h post‐exposure. The increased apoptosis and reduced proliferation in COX‐2−/− mice resulted in about a 50% decrease in epidermal thickness at 24–48 h post‐exposure compared to about a 50% increase in epidermal thickness in WT mice. UVB‐induced cell replication, as measured by BrdU labeling, was reduced in COX‐2−/− compared to WT mice at 24–96 h. However, by 96 h post‐exposure, both WT and COX‐2−/− mice showed epidermal hyperplasia. The data indicate that COX‐2 induction initially protects against the acute sunburn effects of UVB, but that continuous induction of COX‐2 may contribute to skin cancer in chronic UVB exposure.

Disruption of the Mouse Cyclooxygenase 1 Gene

We recently reported the characteristics of mice deficient in cyclooxygenase (COX) 1 (Langenbach et al., 1995) and COX-2 (Morham et al., 1995). This chapter will summarize the known characteristics of COX-1 deficient mice and describe some future studies utilizing these mice which will lead to a better understanding of the physiological functions of the COX’s and their roles in the therapeutic and toxic effects of NSAIDs (non-steroidal anti-inflammatory drugs).

Role of Cyclooxygenase-2 in Exacerbation of Allergen-Induced Airway Remodeling by Multiwalled Carbon Nanotubes

The emergence of nanotechnology has produced a multitude of engineered nanomaterials such as carbon nanotubes (CNTs), and concerns have been raised about their effects on human health, especially for susceptible populations such as individuals with asthma. Multiwalled CNTs (MWCNTs) have been shown to exacerbate ovalbumin (OVA)-induced airway remodeling in mice. Moreover, cyclooxygenase-2 (COX-2) has been described as a protective factor in asthma. We postulated that COX-2-deficient (COX-2(-/-)) mice would be susceptible to MWCNT-induced exacerbations of allergen-induced airway remodeling, including airway inflammation, fibrosis, and mucus-cell metaplasia (i.e., the formation of goblet cells). Wild-type (WT) or COX-2(-/-) mice were sensitized to OVA to induce allergic airway inflammation before a single dose of MWCNTs (4 mg/kg) delivered to the lungs by oropharyngeal aspiration. MWCNTs significantly increased OVA-induced lung inflammation and mucus-cell metaplasia in COX-2(-/-) mice compared with WT mice. However, airway fibrosis after exposure to allergen and MWCNTs was no different between WT and COX-2(-/-) mice. Concentrations of certain prostanoids (prostaglandin D2 and thromboxane B2) were enhanced by OVA or MWCNTs in COX-2(-/-) mice. No differences in COX-1 mRNA concentrations were evident between WT and COX-2(-/-) mice treated with OVA and MWCNTs. Interestingly, MWCNTs significantly enhanced allergen-induced cytokines involved in Th2 (IL-13 and IL-5), Th1 (CXCL10), and Th17 (IL-17A) inflammatory responses in COX-2(-/-) mice, but not in WT mice. We conclude that exacerbations of allergen-induced airway inflammation and mucus-cell metaplasia by MWCNTs are enhanced by deficiencies in COX-2, and are associated with the activation of a mixed Th1/Th2/Th17 immune response.

The chemotherapeutic potential of glycol alkyl ethers: structure-activity studies of nine compounds in a Fischer-rat leukemia transplant model.

SummaryStructure-activity studies with nine glycol alkyl ethers were conducted with a cellular leukemia transplant model in male Fischer rats. This in vivo assay measures the effects of chemical treatment on neoplastic progression in transplant recipients. Chemicals were given ad libitum in the drinking water simultaneously with the transplants and continued throughout the study. In all, 20 million leukemic cells were injected s.c. into syngeneic rats, which after 60 days resulted in a 10-fold increase in relative spleen weights, a 100-fold increase in white blood cell counts, and a 50% reduction in red blood cell (RBC) indices and platelet counts. At this interval, ethylene glycol monomethyl ether (2-ME) given at a dose of 2.5 mg/ml in the drinking water completely eliminated all clinical, morphological, and histopathological evidence of leukemia, whereas the same dose of ethylene glycol monoethyl ether (2-EE) reduced these responses by about 50%. Seven of the glycol ethers were ineffective as anti-leukemic agents, including ethylene glycol, the monopropyl, monobutyl, and monophenyl ethylene glycol ethers, diethylene glycol, and the monomethyl and monoethyl diethylene glycol ethers. 2-ME more than doubled the latency period of leukemia expression and extended survival for at least 210 days. A minimal effective dose for a 50% reduction in the leukemic responses was 0.25 mg/ml 2-ME in the drinking water (15 mg/kg body weight), whereas a 10-fold higher dose of 2-EE was required for equivalent antileukemic activity. In addition, the in vitro exposure of a leukemic spleen mononuclear cell culture to 2-ME caused a dose- and time-dependent reduction in the number of leukemia cells after a single exposure to 1–100 µM concentrations, whereas the 2-ME metabolite, 2-methyoxy-acetic acid, was only half as effective. The two glycol alkyl ethers with demonstrable anti-leukemic activity, 2-ME and 2-EE, also exhibited a favorable efficacy-to-toxicity ratio and should be considered for further development as chemotherapeutic agents.

A proposed COX‐2 and PGE2 receptor interaction in UV‐exposed mouse skin

The cyclooxygenases (COX‐1 and COX‐2) and the prostaglandins (PGs) they generate play a major role in the skins response to sunlight. Sunlight, especially the ultraviolet B (UVB) component, induces COX‐2 and increases PG levels. However, PGs can have both beneficial and adverse cutaneous effects. To elucidate the roles of the COXs and the PGs they generate in response to UVB exposure, experiments with the COX‐1‐ and COX‐2‐deficient mice have provided insight into the specific roles of each isoform. Furthermore, because PGE2 is the major PG produced following UV exposure and PGE2 manifests its biological activity via four membrane receptors (EP1, EP2, EP3, EP4), elucidating contributions of these receptors is essential for understanding the roles of PGs in UVB‐induced effects. In this review, we summarize recent findings from the COX‐deficient mice showing how COX‐2 generated PGE2 acting via the receptors EP2 and EP4 could contribute to short term beneficial, but also contribute to long‐term carcinogenic effects in response to UVB exposure.