• Enzymes
• genetics
• haemato-oncology
• macrophages
• toxicology

Enzyme histochemistry was developed during the first half of the last century to enable morphologists to distinguish between cells of similar appearance in tissue sections. Its use then spread into haematology, and techniques were developed for staining of cells on blood smears. While staining of esterases in tissue sections proved not to be specific or informative enough to be diagnostically useful, this was not the case for white blood cells on peripheral blood and bone marrow smears. Variation of substrate and/or pH provided cell-specific esterase positivity: the use of chloroacetate esters as substrate being specific for non-lymphoid cells, while α-naphthyl acetate or butyrate substrates at specific pH provided positivity confined to cells of monocytic lineage.1–3

In the latter staining procedure the enzyme is visualised in cells by its ability to hydrolyse α-naphthyl acetate or butyrate liberating naphthol groups that are trapped by colourless substrates producing visible compounds. Cytochemical esterase positivity has been demonstrated by isoelectric focusing of various monocytic and non-monocytic cell extracts on polyacrylamide gels to correlate with a specific pattern of isoenzymes occurring only in monocytes.4 5 The restriction of these isoenzymes to monocytes and cells of monocytic lineage was shown by their immunoprecipitation with specific antisera.6

These studies proved that a simple cytochemical staining procedure could specifically identify monocytes, and led to the ability to quantify monocyte contamination of cell suspensions separated from peripheral blood and spun onto slides.7 This enabled correct interpretation of the sheep cell rosetting techniques first used to differentiate between T and B lymphoid cells and their malignant counterparts. This monocyte-specific staining also enabled recognition of the monocytic phenotype of some acute leukaemias and it still contributes to the classification and prognostication of acute leukaemias. In addition, staining of monocyte-specific esterase was incorporated into automated differential white cell counting by Technicon Instruments Corporation (Tarrytown, New York, USA) in their Hemalog-D90 instruments, which were widely used in clinical laboratories until supplanted by instruments using flow technologies.

At the same time as visualisation of this monocyte enzyme, now designated carboxylesterase 1 (Ces1), was becoming clinically useful, knowledge of the interactions between organophosphates (OPs) and Ces1 in vitro and in vivo was accumulating. This has led to recognition of the enzyme as an important entity. It had been known for some time in the biochemical world that carboxylesterases were inhibited by OPs, and this led to their designation as B-esterases, while esterases that hydrolysed OPs were A-esterases, and C-esterases did not interact with OPs at all. Acute exposure to OPs was diagnosed by serum cholinesterase levels and chronic exposure by erythrocyte acetylcholinesterase. Perhaps the first indication that monocyte Ces1 could be affected by exogenous exposure to OPs in vitro and in vivo, and was therefore an indicator of OP exposure, was the demonstration of complete absence of Ces1 staining in blood samples exposed to the air in rooms containing OP-impregnated fly strips.8 Then, a patient who had ingested an OP (malathion) was shown to have complete absence of Ces1 staining in her peripheral blood monocytes until serum cholinesterase had returned to normal levels and the patient recovered,9 and studies of workers exposed to OP compounds demonstrated in vivo inhibition of Ces1 staining.10At the lethal end of OP exposure was the terrorist attack in Tokyo in 1995 when sarin gas was released in the underground. It has been shown that Ces1 stereoselectively binds the nerve agent cyclosarin,11 and now this enzyme is being investigated as a future therapeutic and/or prophylactive agent for nerve gas poisoning, perhaps by a single amino acid substitution in the active site of the enzyme that converts it into a hydrolytic enzyme.12

In vitro studies have suggested another function for Ces1. Inhibition of Ces1 by OPs was shown to be associated with diminished tumour cell killing by monocytes.13 Constitutional deficiency of the enzyme monocyte esterase/Ces1 deficiency (MED) was shown to be associated with diminished ability to lyse K562 cells.14 Low α-naphthyl butyrate esterase activity has been reported in macrophages matured in vitro from peripheral blood monocytes of patients with non-Hodgkin lymphoma. On average, esterase activity was 55% lower at each maturation time than in the normal population similarly investigated,15 and defective phagocytosis and chemotactic activity of monocytes from patients with non-Hodgkin lymphoma has been described.16 These findings contribute to the hypothesis that Ces1 has a role in tumour cell killing or surveillance.

Further evidence to support a possible tumouricidal or tumour surveillance function of Ces1 has been provided by epidemiological study of MED. An approximately 10-fold increased incidence of MED was demonstrated in B lymphoproliferative disease and gastrointestinal carcinoma compared with the incidence in blood donors.17 18 The deficiency has been demonstrated to be a familial characteristic (of autosomal nature),17 19–22 so the increased incidence in these diseases may reflect a susceptibility of MED subjects to the diseases, and substantiate the evidence for a role for the enzyme in tumour surveillance.

Newcombe has hypothesised that the immunological impairment caused by OPs together with their mutagenic effects may interact with viral-mediated lymphoproliferation in lymphomagenesis.23 The in vitro and in vivo evidence of the mutagenic effects of OPs is summarised in Newcombe's paper.23 Subjects with constitutional deficiency of Ces1 may be at additional risk from the low level of dietary OP exposure to which all of us are constantly exposed. This could contribute to the observed increased incidence of MED in patients with B lymphoproliferative disease or gastrointestinal malignancy.17 18

The CES 1/human monocyte/macrophage gene was sequenced in 1991.24 25 Demonstration of CES1 genetic mutations in the consented anonymised samples deposited from patients with MED in the European Collection of Cell Cultures (http://www.hpacultures.org.uk/) would lend further weight to the hitherto cytochemical findings—even the identification of the isoenzymes is dependent on a final esterase stain—and thus to the hypothesis that Ces1 has a function in tumour surveillance/killing. Gene sequencing of CES1 in six patients with MED at the beginning of this century appeared to show a consistent deletion at 52+22∆T in exon 1 of CES1,26 but further studies would be necessary to determine whether the deletion is significant. The characterisation of CES1 has been considerably elucidated since then.27

While further work may be necessary to prove beyond doubt a function for Ces1 in tumour surveillance, it is clear that Ces1 is a major player in the metabolism of many drugs. It has been crystallised in complex with a cocaine analogue,28 and determination of its structure has enabled prediction of drugs that it would specifically metabolise and those that it would partially metabolise.12

It has been demonstrated to play a highly specific role in the metabolism of several therapeutic agents: methylphenidate (MPH),29 heroin, cocaine,28 30 capecitabine31 (an anti-cancer agent), certain ace inhibitors,32 33 and oseltamivir34 (Tamiflu: the anti-influenza drug for treatment of patients with ‘swine flu’). Some of these drugs, for example oseltamivir, capecitabine, heroin and ace inhibitors, are activated by Ces1, suggesting that deficiency of the enzyme might cause them to be relatively ineffective. In contrast MPH is inactivated by Ces1,29 suggesting that lack of Ces1 activity might predispose to toxicity from the uninactivated agent, or indeed that a lower dose might be effective.

There is some evidence to support these two hypotheses. MPH is the drug most commonly used for the treatment of attention deficit hyperactivity disorder. Ces1 metabolises MPH to ritalinic acid, which is inactive. Deficiency of Ces1 activity in plasma (defined by inability to hydrolyse naphthyl ester) has been demonstrated in a subject who showed greatly elevated levels of plasma MPH compared with others given the same dose of MPH.35 Haemodynamic measurements showed this subject to be an outlier for all endpoints assessed, suggesting a potential for the occasionally described adverse cardiovascular events ascribed to MPH. The subject had two CES1 mutations: p.Gly143Glu in exon 4 and a frameshift mutation p.Asp260fs in exon 6. Another recent study demonstrated that a lower dose of MPH was effective in children who, similar to the above subject, had the CES1 mutation Gly143Glu.36 It would be of interest to examine the Ces1 activity of the monocytes of these children. It would also be informative to examine the stored samples from MED subjects in the European Collection of Cell Cultures for this mutation inter alia. A simple test such as Ces1 staining of monocytes that correlated with CES1 mutation and aberrant metabolism of a particular drug would be useful.

It is likely that similar genetic mutations will be found to affect the metabolism of the prodrugs activated by Ces1, rendering ineffective those specifically activated by it. Irinotecan is a drug activated by Ces1.37 38 In a recent relatively small study of patients with colorectal carcinoma who were being treated with irinotecan, the area under the curve of SN-38, the active metabolite of irinotecan, showed a correlation of 0.38 (p=0.013) with monocyte Ces1 activity.39 Irinotecan is also activated to SN-38 by carboxylesterase 2, with some evidence suggesting that this enzyme has greater activity than Ces1 for irinotecan.40 It is not then surprising that the correlation demonstrated in the study described above was relatively low.

The monocyte Ces1 activity of the families of the patients in the study described above was not evaluated, but the familiality of MED demonstrated in the families previously referred to shows that Ces1 levels in monocytes are genetically determined. It is probable that mutations affecting its metabolic activity towards drugs activated by Ces1 will also be shown in time, as has been the case with drugs inactivated by it, for example MPH and Tamiflu. It is reasonable to expect that the effects of Ces1 inactivity will be of greater significance with regard to drugs that are specifically metabolised by Ces1 than those metabolised by more than one enzyme. It is also of interest that a mutant human Ces1, which when expressed in human tumour cells greatly increases their sensitivity to irinotecan in vitro, has been developed with a view to future enzyme/prodrug therapy with irinotecan.41

The irinotecan study referred to above was limited in the number of patients involved and the time points for evaluation of the SN-38 area under the curve. Nevertheless, the correlation with peripheral blood Ces1/monocyte specific esterase level is of interest and could suggest that monocyte Ces1 activity is representative of total body Ces1 activity. Yet Ces1 is universally referred to in pharmacological studies as a liver enzyme. If it is a hepatocyte enzyme it would be foolish to postulate that monocyte Ces1 could represent total Ces1 potential activity. However, the use of in situ hybridisation with digoxigenin-labelled human macrophage serine esterase/CES1 cDNA has demonstrated Ces1 mRNA in monocytes and macrophages only, the latter including the Kupffer cells of the liver.42 The large diffuse Kupffer cell content of the liver and its intimacy with hepatocytes could potentially cause confusion in the attribution of Ces1 activity of liver microsomes to hepatocytes in pharmacological studies. This is an important point to resolve because if Ces1 resides only in the monocyte/macrophage system, determination of peripheral blood monocyte Ces1 activity could become an important predictor of efficacy and/or toxicity of drugs specifically metabolised by Ces1.

There are well-established facts outlined above, as well as some unresolved matters. It is tempting to propose Ces1 as a major player in interaction with external agents, therapeutic or otherwise, and internal adversities such as certain tumours. Whether or to what extent future study supports this view, it is certain that from a simple cytochemical test demonstrating an enzyme activity in the middle of the last century a large body of knowledge has accumulated from a variety of disciplines and shows Ces1 to be a very interesting and important enzyme.