Detecting and Measuring Cyclooxygenases

by Kirk Maxey, M.D. and Jennifer Johnson

Cyclooxygenase (COX, also called Prostaglandin H Synthase or PGHS) is a bifunctional enzyme exhibiting both cyclooxygenase and peroxidase activities. The cyclooxygenase component converts arachidonic acid to a hydroperoxy endoperoxide (PGG₂), and the peroxidase component reduces the hydroperoxide to the corresponding alcohol (PGH₂), which is the precursor of prostaglandins, thromboxanes, and prostacyclins (see Figure 1 below).^1,2 COX enzymes exist as homodimers embedded in the membrane of the nucleus and endoplasmic reticulum.

It is now well established that there are two distinct isoforms of cyclooxygenase. Cyclooxygenase-1 (COX-1) is constitutively expressed in a variety of cell types. COX-1 produces prostaglandins that serve protective functions such as sodium and water resorption, gastric cytoprotection, and vascular homeostasis. A variety of mitogenic stimuli such as phorbol esters, lipopolysaccharides, and cytokines lead to the induced expression of a second isoform of cyclooxygenase, cyclooxygenase-2 (COX-2). COX-2 is responsible for the biosynthesis of prostaglandins under acute inflammatory conditions, in certain cancers, and in the brain.³ The last decade saw an enormous expansion in research relating to possible roles for COX enzymes in cancer, Alzheimer’s disease, and inflammation, which have sparked major new efforts in drug discovery. Various methods can be employed to characterize the expression, localization, activity, and inhibitory profile of each COX isoform.

mRNA Quantitation & Localization

Although COX-1 is generally described as constitutively expressed, this is actually an over-simplification. COX-1 expression is regulated developmentally and in response to a variety of other stimuli.^4-7 Northern blot analysis reveals that the mRNA for human COX-1 is approximately 2.9 kb in length and is expressed in nearly all tissues.^4,8 In contrast, COX-2 mRNA is rarely expressed in quiescent cells, but is expressed in a wide range of cell types following the appropriate stimulation.⁸ Human COX-2 mRNA is approximately 4.4 kb in length.^9,10 Cayman offers a wide range of products to allow mRNA quantitation and localization. Traditional probes can be used for Northern blot analysis. Probe templates are available in both sense and antisense orientation to allow detection of mRNA by both RNase protection assay and in situ hybridization, with all of the appropriate controls (see Table 1).

Quantitation & Localization of COX Protein

COX-1 and COX-2 have subunit molecular weights of 70 and 72 kDa, respectively, when denatured in sodium dodecyl sulfate and analyzed by polyacrylamide gel electrophoresis (SDS-PAGE). Human COX-1 and COX-2 polypeptides share 68% primary sequence identity. Interestingly, COX-2 contains a unique 18-amino acid insertion in the C-terminal region, which contains a putative fourth glycosylation site.¹¹ This unique region allows for the generation of specific COX-2 antibodies. Cayman has developed a variety of monoclonal and polyclonal antibodies, which can be used for immunoblotting (Western blotting), and immunohisto(cyto) chemistry. A description of each antibody, including cross-reactivities, is outlined in the table below.

Cyclooxygenase Activity Measurements

Finding a COX-2 specific inhibitor that exhibits anti-inflammatory and analgesic effects without the adverse effects that characterize the currently available nonsteroidal anti-inflammatory drugs (NSAIDs) accounts for the majority of COX research. At the biochemical level, COX inhibitors are usually characterized by comparing their in vitro inhibitory effects on COX activity. Several methods have been described for the determination of COX activity.

A) Polarographic (Oxygraph)

Cyclooxygenase activity can be measured by monitoring oxygen consumption using an oxygraph equipped with an oxygen electrode. The reaction mixture consists of a Tris buffer (pH 8.0) containing EDTA, hematin, phenol, and COX. The reaction is initiated with arachidonate and the rate of oxygen consumption is charted on graph paper.

Advantages: Accurate (direct consumption of O₂ is measured); continuous; measures cyclooxygenase activity directly; excellent for detailed inhibitor/enzyme mechanistic studies.

Disadvantages: Not adaptable to high-throughput assay; lack of instrumentation in most labs; typically requires ~50 units of enzyme.

B) Prostaglandin Detection

Prostaglandins produced in the cyclooxygenase reaction can be quantified via enzyme immunoassay (EIA). The reaction mixture contains a Tris buffer (pH 8.0) containing EDTA, hematin, phenol, and COX. The reaction is initiated with arachidonic acid, incubated for a specific time at a specific temperature, acidified, and saturated stannous chloride is added to reduce PGH₂ to the more stable prostaglandin PGF_2α. Prostaglandins are then quantified by EIA.

Advantages: Excellent tool for general inhibitor screening; sensitive (requires <1 unit of enzyme).

Disadvantages: Sample may require purification (depends on enzyme source); single time-point assay.

C) Peroxidase

Cyclooxygenases are bifunctional enzymes exhibiting both cyclooxygenase and peroxidase activities. The second reaction catalyzed by the peroxidase component is the reduction of the hydroperoxy endoperoxide (PGG₂) to the corresponding alcohol (PGH₂). This reaction requires a second, reducing substrate which is oxidized as the hydroperoxide is reduced. Phenol is the usual reducing substrate for COX activity assays, but it may be replaced by other oxidizable aromatics. Selected reducing substrates generate a chromophore when oxidized, detectable by UV/Vis or luminescent detection. This gives rise to at least three general methods for determining the peroxidase activity of cyclooxygenases:

1)

Colorimetric determination of peroxidase can be determined by following the oxidation of N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) using hydrogen peroxide or arachidonic acid as the substrate.¹² The reaction mixture includes hematin, TMPD, and COX. The reaction is initiated with substrate (hydrogen peroxide or arachidonic acid) and an increase in absorbance at 611 nm is recorded on a spectrophotometer. The peroxidase activity is calculated based on an extinction coefficient of 12,200 M^-1cm^-1 and a stoichiometry of 2 mol of TMPD oxidized per mole of hydroperoxide reduced.¹³

Advantages: Can be performed on UV/Vis spectrophotmeter or can be adapted to a microplate assay; excellent tool for general inhibitor screening.

Disadvantages: Does not measure cyclooxygenase activity directly; antioxidants will interfere with assay.

2)

The peroxidase activity of cylooxygenases can generate luminescence in the presence of luminol as the reducing substrate.¹⁴ The reaction contains hematin, luminol, and COX, and is initiated with arachidonic acid. Luminescence is recorded on a luminometer.

Advantages: Reactions can be performed in a test tube or microplate luminometer; excellent tool for general inhibitor screening; extremely sensitive, allows conservation of scarce recombinant COX enzyme.

Disadvantages: Does not measure cyclooxygenase activity directly; luminometers are expensive and not available in all labs; antioxidants will interfere with assay.

3)

Peroxidase activity can be determined directly by following the reduction of 5-phenyl-4-pentyl hydroperoxide (PPHP) to the corresponding alcohol 5-phenyl-4-pentenyl alcohol (PPA).¹⁵ The reaction mixture consists of a Tris buffer (pH 8.0) containing EDTA, phenol, and COX. The reaction is initiated with PPHP, incubated for two minutes, reactions are acidified, and PPHP/PPA is extracted with a C₁₈ reverse-phase cartridge. The filtered eluent is used directly for HPLC analysis on a reverse-phase C₁₈ column. The eluent is monitored at 252 nm. PPHP and PPA concentrations are calculated by reference to an internal standard.¹⁵

Advantages: Directly measures peroxidase activity; can standardize assay using internal controls.

Disadvantages: Lack of instrumentation in some labs (HPLC); does not measure cyclooxygenase activity; not adaptable to high-throughput assay; samples have to be purified; not very sensitive; single time-point assay (linearity needs to be established for assay conditions).

Beyond initial pharmacological testing in animals, the best indication of COX selectivity in humans is provided by the ex vivo whole blood assay.¹⁶ In this assay, PGE₂ levels in lipopolysaccharide-challenged human whole blood and thromboxane B₂ levels following blood coagulation are measured by EIA as biochemical indexes for COX-2 and COX-1 activity, respectively. This assay can be used to assess the biochemical efficacy of selective COX-2 inhibitors in clinical trials.

References

1. Nugteren, D.H. and Hazelhof, E. Isolation and properties of intermediates in prostaglandin biosynthesis. Biochim. Biophys. Acta 326, 448-461 (1973).
2. Hamberg, M. and Samuelsson, B. Detection and isolation of an endoperoxide intermediate in prostaglandin biosynthesis. Proc. Natl. Acad. Sci. USA 70, 899-903 (1973).
3. Xie, W., Chipman, J.G., Robertson, D.L., et al. Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc. Natl. Acad. Sci. USA 88, 2692-2696 (1991).
4. Brannon, T.S., North, A.J., Wells, L.B., et al. Prostacyclin synthesis in ovine pulmonary artery is developmentally regulated by changes in cyclooxygenase-l gene expression. J. Clin. Invest. 93, 2230-2235 (1994).
5. Funk, C.D., Funk, L.B., Kennedy, M.E., et al. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment. FASEB J. 5, 2304-2312 (1991).
6. Samet, J.M., Fasano, M.B., Fonleh, A.N., et al. Selective induction of prostaglandin G/H synthase I by stem cell factor and dexamethasone in mast cells. J. Biol. Chem. 270, 8044-8049 (1995).
7. Hla, T. and Maciag, T. Cyclooxygenase gene expression is down-regulated by heparin-binding (acidic fibroblast) growth factor-1 in human endothelial cells. J. Biol. Chem. 266, 24059-24063 (1991).
8. Smith, W.L., Garavito, R.M., and DeWitt, D.L. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J. Biol. Chem. 271, 33157-33160 (1996).
9. Hla, T. and Neilson, K. Human cyclooxygenase-2 cDNA. Proc. Natl. Acad. Sci. USA 89, 7384-7388 (1992).
10. Jones, D.A., Carlton, D.P., McIntyre, T.M., et al. Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines. J. Biol. Chem. 268, 9049-9054 (1993).
11. Appleby, S.B., Ristimäki, A., Neilson, K., et al. Structure of the human cyclo-oxygenase-2 gene. Biochem. J. 302, 723-727 (1994).
12. Kulmacz, R.J. and Lands, W.E.M. Requirements for hydroperoxide by the cyclooxygenase and peroxidase activities of prostaglandin H synthase. Prostaglandins 25, 531-540 (1983).
13. Kulmacz, R.J. Prostaglandin G₂ levels during reaction of prostaglandin H synthase with arachidonic acid. Prostaglandins 34, 225-240 (1987).
14. Forghani, F., Ouellet, M., Keen, S., et al.. Analysis of prostaglandin G/H synthase-2 inhibition using peroxidase-induced luminal luminescence. Anal. Biochem. 264, 216-221 (1998).
15. Markey, C.M., Alward, A., Weller, P.E., et al. Quantitative studies of hydroperoxide reduction by prostaglandin H synthase: Reducing substrate specificity and the relationship of peroxidase to cyclooxygenase activities. J. Biol. Chem. 262, 6266-6279 (1987).
16. Brideau, C., Kargman, S., Liu, S., et al. A human whole blood assay for clinical evaluation of biochemical efficacy of cyclooxygenase inhibitors. Inflamm. Res. 45, 68-74 (1996).

The Use of Biological Markers to Document Environmental Endocrine Disruption

Endocrine disruption is a term which refers to the harm caused by incidental exposure to environmental pesticides, industrial chemicals, or synthetic hormones which specifically target and interact with the normal endocrine hormonal system of the exposed organism. Although endocrine disruption (or endocrine modulation) only recently emerged as a major issue in terms of science, public concern, and regulatory policies, the first sign of chemically induced alterations of endocrine function dates back several decades. It was seen already in the late 1930's that several synthetic chemicals were able to bind to estrogen receptors in the rat uterus.1 In 1962, Rachel Carson’s book “Silent Spring” spurred a global interest in the effects of DDT on eggshell thinning in birds, and in the early 1970's, the presence of imposex (development of male reproductive organs in females) in different species of marine neogastropod molluscs was correlated to exposure to tributyltin (TBT), an antifouling agent used in paint.^2,3 In recent investigations, researchers have reported abnormal sexual development and sex steroid levels in alligators exposed to organochlorine pesticides, whereas fish exposed to effluents from municipal and industrial activities have shown signs of both feminization, masculinization, and intersex conditions.^4,5 Although controversial, an increase in the incidence of human testicular abnormalities, testicular cancer, and a decrease in sperm quality (sperm counts, ejaculate volume, and sperm motility) of normal healthy men over the last 50 years, has recently been proposed to be caused by the increasing exposure to environmental contaminants acting as endocrine disrupters.⁶

Of this group of endocrine disrupters, considerable attention has been given to the environmental estrogens (see Figure 2). These xenoestrogens or estrogen mimics are able to act as estrogen agonists by inducing estrogenic responses in animals. A common feature of these compounds is their inherent ability to bind to and activate the nuclear estrogen receptor (ER) and stimulate the transcription of estrogen sensitive genes. While detrimental to normal reproduction and development, ER-mediated transcription of estrogen sensitive genes has proved invaluable for the development of biological markers (biomarkers) for estrogenic effects of chemicals. For instance, the synthesis of the yolk precursor protein vitellogenin (Vtg) and the eggshell zona radiata proteins (Zrp) take place in the liver of female egglaying fish under the stimulation of low concentrations of endogenous estrogen. Male fish and juvenile fish, which exhibit extremely low levels of circulating estrogens, do not produce appreciable levels of either Vtg or Zrp. However, these fish express the hepatic ER and the genetic machinery required for protein synthesis, and are thus capable of producing measurable levels of both Zrp and Vtg when exposed to exogenous estrogens (see Figure 3). Induction of these female proteins in male and juvenile fish has therefore been proposed to be sensitive biomarkers for exposure to environmental estrogens and employed with success for chemical screening and environmental monitoring of effluents and natural waters.⁵

In a recent initiative from an Organization for Economic Cooperation and Development (OECD) Task Force for the development and harmonization of Endocrine Disrupter Testing and Assessment (EDTA), Vtg was acknowledged as a useful biomarker for estrogenic and anti-estrogenic effects in fish. The EDTA endorsed previous recommendations to include Vtg as an endpoint in a short term fish-screening assay for endocrine disrupting chemicals (EDCs) with estrogenic properties. In their proposal, which will probably have major implications for the future design of screening tests for EDCs, they suggest that the short term fish tests should be designed to be applicable to different species, in particular zebrafish (Danio rerio), fathead minnow (Pimephales promelas), carp (Cyprinus carpio), Japanese medaka (Oryzias latipes), and rainbow trout (Oncorhynchus mykiss).

The use of the yolk precursor protein vitellogenin (Vtg) as a biomarker for estrogenic effects in fish has greatly improved the screening of chemicals and monitoring of natural waters for estrogen mimics. The reason for some of the success must be accredited to the sensitivity and specificity of the protein induction in fish. Importantly, the induction of Vtg is a direct physiological response to an exposure, rather than the mere presence of a chemical, and will thus take into account both bioavailability and the complex toxicokinetics and toxicodynamics that occur in animals. Furthermore, the use of biological endpoints in the risk assessment will ensure that effects of multiple chemicals with identical mechanism of action as well as temporal variation in exposure will be integrated. Finally, immunological assays (Enzyme-Linked Immuno Sorbent Assay, ELISA) employed for the analysis require low-level analytical technology, have high sample throughput and are inexpensive compared to traditional chemical analysis. The advent of commercially available, sensitive, specific, simple, and robust ELISA assays for Vtg in the different OECD test species of fish, will greatly enhance the usefulness of this biomarker in the chemical screening programs as well as in environmental monitoring.

Cayman now offers, in collaboration with Biosense Laboratories, AS, a broad line of assays and antibodies for use in the detection of environmental endocrine disruptors.

Additional Figure

References

1. Dodds, E.C. and Lawson, W. Molecular structure in relation to oestrogenic activity. Compounds without a phenanthrene nucleus. Proc. Roy. Soc. Biol. 125, 222-232 (1938).
2. Carson, R. "Silent Spring." Fawcett Crest Book, Greenwich, USA (1962).
3. Babler, S.J.M. The occurrence of a penis -like outgrowth behind the right tentacle in spent female of Nucella Lapillus (L.). Proc. Malacol. Soc. London. 39, 231-233 (1970).
4. Guillette, L.J., Jr., Gross, T.S., Masson, G.R., et al. Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environ. Health Perspect. 102, 680-688 (1994).
5. Arukwe, A. and Goksoyr, A. Xenobiotics, xenoestrogens and reproduction disturbances in fish. SARSIA 83, 225-241 (1998).
6. Toppari, J., Larsen, J.C., Christiansen, P., et al. Male reproductive health and environmental xenoestrogens. Environ. Health Perspect. 104, 741-803 (1996).