An enzyme trafficking defect in two patients with primary hyperoxaluria type 1: peroxisomal alanine/glyoxylate aminotransferase rerouted to mitochondria

Most patients with the autosomal recessive disease primary hyperoxaluria type 1 (PH1) have a complete deficiency of alanine/glyoxylate aminotransferase (AGT) enzyme activity and immunoreactive protein. However a few possess significant residual activity and protein. In normal human liver, AGT is entirely peroxisomal, whereas it is entirely mitochondrial in carnivores, and both peroxisomal and mitochondrial in rodents. Using the techniques of isopycnic sucrose and Percoll density gradient centrifugation and quantitative protein A-gold immunoelectron microscopy, we have found that in two PH1 patients, possessing 9 and 27% residual AGT activity, both the enzyme activity and immunoreactive protein were largely mitochondrial and not peroxisomal. In addition, these individuals were more severely affected than expected from the levels of their residual AGT activity. In these patients, the PH1 appears to be due, at least in part, to a unique trafficking defect, in which peroxisomal AGT is diverted to the mitochondria. To our knowledge, this is the first example of a genetic disease caused by such interorganellar rerouting.     

Immunological heterogeneity of hepatic alanine:glyoxylate aminotransferase in primary hyperoxaluria type 1

Immunoblotting of human liver sonicates, after SDS-polyacrylamide gel electrophoresis, demonstrated the presence of a 40 kDa protein, corresponding to the subunit of alanine:glyoxylate aminotransferase, in six controls and three patients with primary hyperoxaluria type 1 (peroxisomal alanine:glyoxylate aminotransferase deficiency). This immunoreactive 40 kDa protein was absent in a further nine patients. Subcellular fractionation of patients' livers showed that the 40 kDa protein, when present, was located mainly in the peroxisomes. In a heterozygote liver, the 40 kDa protein was also mainly peroxisomal and paralleled the distribution of alanine:glyoxylate aminotransferase activity.     

Enzymological and mutational analysis of a complex primary hyperoxaluria type 1 phenotype involving alanine:Glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting and intraperoxisomal aggregation

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disease caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). Three unrelated PH1 patients, who possess a novel complex phenotype, are described. At the enzymological level, this phenotype is characterized by a complete, or nearly complete, absence of AGT catalytic activity and reduced AGT immunoreactivity. Unlike normal individuals in whom the AGT is confined to the peroxisomal matrix, the immunoreactive AGT in these three patients was distributed approximately equally between the peroxisomes and mitochondria. The peroxisomal AGT appeared to be aggregated into amorphous core-like structures in which no other peroxisomal enzymes could be identified. Mutational analysis of the AGT gene showed that two of the three patients were compound heterozygotes for two previously unrecognized point mutations which caused Gly41→Arg and Phe152→Iso amino acid substitutions. The third patient was shown to be a compound heterozygote for the Gly41→Arg mutation and a previously recognized Gly170→Arg mutation. All three patients were homozygous for the Pro11→Leu polymorphism that had been found previously with a high allelic frequency in normal populations. It is suggested that the Phe152→Iso and Gly170→Arg substitutions, which are only eighteen residues apart and located in the same highly conserved internal region of 58 amino acids, might be involved in the inhibition of peroxisomal targeting and/or import of AGT and, in combination with the Pro11→Leu polymorphism, be responsible for its aberrant mitochondrial compartmentalization. On the other hand, the Gly41→Arg substitution, either in combination with the Pro11→Leu polymorphism or by itself, is predicted to be responsible for the intraperoxisomal aggregation of the AGT protein.     

Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1

Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway. Although the COOH-terminal KKL of human AGT was shown to be necessary for its peroxisomal import, this tripeptide was unable to direct the peroxisomal import of the bona fide peroxisomal protein firefly luciferase or the reporter protein bacterial chloramphenicol acetyltransferase. An ill-defined region immediately upstream of the COOH-terminal KKL was also found to be necessary for the peroxisomal import of AGT, but again this region was found to be insufficient to direct the peroxisomal import of chloramphenicol acetyltransferase. Substitution of the COOH-terminal KKL of human AGT by the COOH-terminal tripeptides found in the AGTs of other mammalian species (SQL, NKL), the prototypical PTS1 (SKL), or the glycosomal PTS1 (SSL) also allowed peroxisomal targeting, showing that the allowable PTS1 motif in AGT is considerably more degenerate than, or at least very different from, that acceptable in luciferase. AGT possessing the two amino acid substitutions responsible for its mistargeting in PH1 (i.e., Pro11-- >Leu and Gly170-->Arg) was targeted mainly to the mitochondria. However, AGTs possessing each amino acid substitution on its own were targeted normally to the peroxisomes. This suggests that Gly170-->Arg- mediated increased functional efficiency of the otherwise weak mitochondrial targeting sequence (generated by the Pro11-->Leu polymorphism) is not due to interference with the peroxisomal targeting or import of AGT.     

ATP-dependent degradation of a mutant serine: pyruvate/alanine:glyoxylate aminotransferase in a primary hyperoxaluria type 1 case

Primary hyperoxaluria type 1 (PH 1), an inborn error of glyoxylate metabolism characterized by excessive synthesis of oxalate and glycolate, is caused by a defect in serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT). This enzyme is peroxisomal in human liver. Recently, we cloned SPT/AGT-cDNA from a PH 1 case, and demonstrated a point mutation of T to C in the coding region of the SPT/AGT gene encoding a Ser to Pro substitution at residue 205 (Nishiyama, K., T. Funai, R. Katafuchi, F. Hattori, K. Onoyama, and A. Ichiyama. 1991. Biochem. Biophys. Res. Commun. 176:1093-1099). In the liver of this patient, SPT/AGT was very low with respect to not only activity but also protein detectable on Western blot and immunoprecipitation analyses. Immunocytochemically detectable SPT/AGT labeling was also low, although it was detected predominantly in peroxisomes. On the other hand, the level of translatable SPT/AGT-mRNA was higher than normal, indicating that SPT/AGT had been synthesized in the patient's liver at least as effectively as in normal liver. Rapid degradation of the mutant SPT/AGT was then demonstrated in transfected COS cells and transformed Escherichia coli, accounting for the low level of immunodetectable mutant SPT/AGT in the patient's liver. The mutant SPT/AGT was also degraded much faster than normal in an in vitro system with a rabbit reticulocyte extract, and the degradation in vitro was ATP dependent. These results indicate that a single amino acid substitution in SPT/AGT found in the PH1 case leads to a reduced half- life of this protein. It appears that the mutant SPT/AGT is recognized in cells as an abnormal protein to be eliminated by degradation.     

Primary hyperoxaluria type 1: AGT mistargeting highlights the fundamental differences between the peroxisomal and mitochondrial protein import pathways

Primary hyperoxaluria type 1 (PH1) is an atypical peroxisomal disorder, as befits a deficiency of alanine:glyoxylate aminotransferase (AGT), which is itself an atypical peroxisomal enzyme. PH1 is characterized by excessive synthesis and excretion of the metabolic end-product oxalate and the progressive accumulation of insoluble calcium oxalate in the kidney and urinary tract. Disease in many patients is caused by a unique protein trafficking defect in which AGT is mistargeted from peroxisomes to mitochondria, where it is metabolically ineffectual, despite remaining catalytically active. Although the peroxisomal import of human AGT is dependent upon the PTS1 import receptor PEX5p, its PTS1 is exquisitely specific for mammalian AGT, suggesting the presence of additional peroxisomal targeting information elsewhere in the AGT molecule. This and many other functional peculiarities of AGT are probably a consequence of its rather chequered evolutionary history, during which much of its time has been spent being a mitochondrial, rather than a peroxisomal, enzyme. Analysis of the molecular basis of AGT mistargeting in PH1 has thrown into sharp relief some of the fundamental differences between the requirements of the peroxisomal and mitochondrial protein import pathways, particularly the properties of peroxisomal and mitochondrial matrix targeting sequences and the different conformational limitations placed upon importable cargos.     

Primary hyperoxaluria type 1 in Japan

Glyoxylate is an immediate precursor of oxalate, but in its metabolism the conversion into glycine catalyzed by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) appears to be the main route. When SPT/AGT is missing as in the case of primary hyperoxaluria type 1 (PH1) more glyoxylate is used for the oxalate production, resulting in calcium oxalate urolithiasis and finally systemic oxalosis. SPT/AGT is a unique enzyme of species-specific dual organelle localization; it is located largely in mitochondria in carnivores and entirely in peroxisomes in herbivores and man. For herbivores, the peroxisomal localization of SPT/AGT is indispensable to avoid massive production of oxalate, probably because liver peroxisomes are the main site of glyoxylate production from glycolate, and plants contain glycolate much more than animal tissues. Recently, we took charge of laboratory examination for 8 cases of primary hyperoxaluria in Japan, and felt that symptoms of some of the Japanese PH1 patients are apparently milder than those of Western patients. The reason of this is not clear, but from the above mentioned seemingly indispensable association of grass-eating with the peroxisomal localization of SPT/AGT it may be related, at least in part, to the food habit of Japanese, especially that of old generation, that they prefer boiled greens rather than frying or raw vegetables.     

TAT-Mediated Delivery of Human Alanine:Glyoxylate Aminotransferase in a Cellular Model of Primary Hyperoxaluria Type I

Defects in liver peroxisomal alanine:glyoxylate aminotransferase (AGT), as a consequence of inherited mutations on the AGXT gene, lead to primary hyperoxaluria type I (PH1), a rare metabolic disorder characterized by the formation of calcium oxalate stones at first in the urinary tract and then in the whole body. The curative treatments currently available for PH1 are pyridoxine therapy, effective in only 10–30 % of the patients, and liver transplantation, an invasive procedure with potentially serious complications. A valid therapeutic option for PH1 patients would be the development of an enzyme administration therapy. However, the exogenous administration of the missing AGT would require the crossing of the plasma membrane to deliver the protein to liver peroxisomes. In this study, we constructed, purified and characterized the fusion protein of AGT with the membrane-penetrating Tat peptide (Tat-AGT). Although Tat-AGT shows subtle active site conformational changes as compared with untagged AGT, it retains a significant transaminase activity. Western-blot analyses, enzymatic assays and immunofluorescence studies show that active Tat-AGT can be successfully delivered to a mammalian cellular model of PH1 consisting of chinese hamster ovary cells expressing glycolate oxidase (CHO-GO), whereas untagged AGT cannot. Moreover, the intracellular transduced Tat-AGT makes CHO-GO cells able to detoxify endogenously produced glyoxylate to an extent similar to that of CHO-GO cells stably expressing AGT. Altogether, these results show that the Tat peptide is capable of delivering a functional AGT to mammalian cells, thus paving the way for the possibility to use Tat-AGT as an enzyme replacement therapy to counteract PH1.     

Inhibition of alanine:glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1

Peroxisome-to-mitochondrion mistargeting of the homodimeric enzyme alanine:glyoxylate aminotransferase 1 (AGT) in the autosomal recessive disease primary hyperoxaluria type 1 (PH1) is associated with the combined presence of a normally occurring Pro(11)Leu polymorphism and a PH1-specific Gly170Arg mutation. The former leads to the formation of a novel NH2-terminal mitochondrial targeting sequence (MTS), which although sufficient to direct the import of in vitro-translated AGT into isolated mitochondria, requires the additional presence of the Gly170Arg mutation to function efficiently in whole cells. The role of this mutation in the mistargeting phenomenon has remained elusive. It does not interfere with the peroxisomal targeting or import of AGT. In the present study, we have investigated the role of the Gly170Arg mutation in AGT mistargeting. In addition, our studies have led us to examine the relationship between the oligomeric status of AGT and the peroxisomal and mitochondrial import processes. The results obtained show that in vitro-translated AGT rapidly forms dimers that do not readily exchange subunits. Although the presence of the Pro(11)Leu or Gly170Arg substitutions alone had no effect on dimerization, their combined presence abolished homodimerization in vitro. However, AGT containing both substitutions was still able to form heterodimers in vitro with either normal AGT or AGT containing either substitution alone. Expression of various combinations of normal and mutant, as well as epitope-tagged and untagged forms of AGT in whole cells showed that normal AGT rapidly dimerizes in the cytosol and is imported into peroxisomes as a dimer. This dimerization prevents mitochondrial import, even when the AGT possesses an MTS generated by the Pro(11)Leu substitution. The additional presence of the Gly170Arg substitution impairs dimerization sufficiently to allow mitochondrial import. Pharmacological inhibition of mitochondrial import allows AGT containing both substitutions to be imported into peroxisomes efficiently, showing that AGT dimerization is not a prerequisite for peroxisomal import.     

A glycine-to-glutamate substitution abolishes alanine:glyoxylate aminotransferase catalytic activity in a subset of patients with primary hyperoxaluria type 1.

We have synthesized and sequenced alanine:glyoxylate aminotransferase (AGT; HGMW-approved symbol for the gene--AGXT) cDNA from the liver of a primary hyperoxaluria type 1 (PH1) patient who had normal levels of hepatic peroxisomal immunoreactive AGT protein, but no AGT catalytic activity. This revealed the presence of a single point mutation (G----A at cDNA nucleotide 367), which is predicted to cause a glycine-to-glutamate substitution at residue 82 of the AGT protein. This mutation is located in exon 2 of the AGT gene and leads to the loss of an AvaI restriction site. Exon 2-specific PCR followed by AvaI digestion showed that this patient was homozygous for this mutation. In addition, three other PH1 patients, one related to and two unrelated to, but with enzymological phenotype similar to that of the first patient, were also shown to be homozygous for the mutation. However, one other phenotypically similar PH1 patient was shown to lack this mutation. The mechanism by which the glycine-to-glutamate substitution at residue 82 causes loss of catalytic activity remains to be resolved. However, the protein sequence in this region is highly conserved between different mammals, and the substitution at residue 82 is predicted to cause significant local structural alterations.     

Flux of the L-serine metabolism in rabbit, human, and dog livers. Substantial contributions of both mitochondrial and peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase.

L-Serine metabolism in rabbit, dog, and human livers was investigated, focusing on the relative contributions of the three pathways, one initiated by serine dehydratase, another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Under quasi-physiological in vitro conditions (1 mM L-serine and 0.25 mM pyruvate), flux through serine dehydratase accounted for only traces, and that through SPT/AGT substantially contributed no matter whether the enzyme was located in peroxisomes (rabbit and human) or largely in mitochondria (dog). As for flux through serine hydroxymethyltransferase and GCS, the conversion of serine to glycine occurred fairly rapidly, followed by GCS-mediated slow decarboxylation of the accumulated glycine. The flux through GCS was relatively high in the dog and low in the rabbit, and only in the dog was it comparable with that through SPT/AGT. An in vivo experiment with L-[3-3H,14C]serine as the substrate indicated that in rabbit liver, gluconeogenesis from L-serine proceeds mainly via hydroxypyruvate. Because an important role in the conversion of glyoxylate to glycine has been assigned to peroxisomal SPT/AGT from the studies on primary hyperoxaluria type 1, these results suggest that SPT/AGT in this organelle plays dual roles in the metabolism of glyoxylate and serine.     

Identification of mutations associated with peroxisome-to-mitochondrion mistargeting of alanine/glyoxylate aminotransferase in primary hyperoxaluria type 1

We have previously shown that in some patients with primary hyperoxaluria type 1 (PH1), disease is associated with mistargeting of the normally peroxisomal enzyme alanine/glyoxylate aminotransferase (AGT) to mitochondria (Danpure, C.J., P.J. Cooper, P.J. Wise, and P.R. Jennings. J. Cell Biol. 108:1345-1352). We have synthesized, amplified, cloned, and sequenced AGT cDNA from a PH1 patient with mitochondrial AGT (mAGT). This identified three point mutations that cause amino acid substitutions in the predicted AGT protein sequence. Using PCR and allele-specific oligonucleotide hybridization, a range of PH1 patients and controls were screened for these mutations. This revealed that all eight PH1 patients with mAGT carried at least one allele with the same three mutations. Two were homozygous for this allele and six were heterozygous. In at least three of the heterozygotes, it appeared that only the mutant allele was expressed. All three mutations were absent from PH1 patients lacking mAGT. One mutation encoding a Gly----Arg substitution at residue 170 was not found in any of the control individuals. However, the other two mutations, encoding Pro----Leu and Ile----Met substitutions at residues 11 and 340, respectively, cosegregated in the normal population at an allelic frequency of 5-10%. In an individual homozygous for this allele (substitutions at residues 11 and 340) only a small proportion of AGT appeared to be rerouted to mitochondria. It is suggested that the substitution at residue 11 generates an amphiphilic alpha-helix with characteristics similar to recognized mitochondrial targeting sequences, the full functional expression of which is dependent upon coexpression of the substitution at residue 170, which may induce defective peroxisomal import.     

Molecular aetiology of primary hyperoxaluria and its implications for clinical management

The primary hyperoxalurias type 1 (PH1) and type 2 (PH2) are autosomal recessive calcium oxalate kidney stone diseases caused by deficiencies of the metabolic enzymes alanine:glyoxylate aminotransferase (AGT) and glyoxylate/hydroxypyruvate reductase (GR/HPR), respectively. Over 50 mutations have been identified in the AGXT gene (encoding AGT) in PH1, associated with a wide variety of effects on AGT, including loss of catalytic activity, aggregation, accelerated degradation, and peroxisome-to-mitochondrion mistargeting. Some of these mutations segregate and interact synergistically with a common polymorphism. Over a dozen mutations have been found in the GRHPR gene (encoding GR/HPR) in PH2, all associated with complete loss of glyoxylate reductase enzyme activity and immunoreactive protein. The crystal structure of human AGT, but not human GR/HPR, has been solved, allowing the effects of many of the mutations in PH1 to be rationalised in structural terms. Detailed analysis of the molecular aetiology of PH1 and PH2 has led to significant improvements in all aspects of their clinical management. Enzyme replacement therapy by liver transplantation can provide a metabolic cure for PH1, but it has yet to be tried for PH2. New treatments that aim to counter the effects of specific mutations on the properties of the enzymes could be feasible in the not-too-distant future.     

Allele-specific Characterization of Alanine: Glyoxylate Aminotransferase Variants Associated with Primary Hyperoxaluria

Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive kidney stone disease caused by deficiency of the peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT), which is involved in glyoxylate detoxification. Over 75 different missense mutations in AGT have been found associated with PH1. While some of the mutations have been found to affect enzyme activity, stability, and/or localization, approximately half of these mutations are completely uncharacterized. In this study, we sought to systematically characterize AGT missense mutations associated with PH1. To facilitate analysis, we used two high-throughput yeast-based assays: one that assesses AGT specific activity, and one that assesses protein stability. Approximately 30% of PH1-associated missense mutations are found in conjunction with a minor allele polymorphic variant, which can interact to elicit complex effects on protein stability and trafficking. To better understand this allele interaction, we functionally characterized each of 34 mutants on both the major (wild-type) and minor allele backgrounds, identifying mutations that synergize with the minor allele. We classify these mutants into four distinct categories depending on activity/stability results in the different alleles. Twelve mutants were found to display reduced activity in combination with the minor allele, compared with the major allele background. When mapped on the AGT dimer structure, these mutants reveal localized regions of the protein that appear particularly sensitive to interactions with the minor allele variant. While the majority of the deleterious effects on activity in the minor allele can be attributed to synergistic interaction affecting protein stability, we identify one mutation, E274D, that appears to specifically affect activity when in combination with the minor allele.     

Alanine:glyoxylate aminotransferase peroxisome-to-mitochondrion mistargeting in human hereditary kidney stone disease

The pyridoxal-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT) is mistargeted from peroxisomes to mitochondria in patients with the hereditary kidney stone disease primary hyperoxaluria type 1 (PH1) due to the synergistic interaction between a common Pro(11)Leu polymorphism and a PH1-specific Gly(170)Arg mutation. The kinetic partitioning of newly synthesised AGT between peroxisomes and mitochondria is determined by the combined effects of (1) the generation of cryptic mitochondrial targeting information, and (2) the inhibition of AGT dimerization. The crystal structure of AGT has recently been solved, allowing the effects of the various polymorphisms and mutations to be rationalised in terms of AGT's three-dimensional conformation. Procedures that increase dimer stability and/or increase the rate of dimer formation have potential in the formulation of novel strategies to treat this otherwise intractable life-threatening disease.     

Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana

Leaf peroxisomes are present in greening cotyledons and contain enzymes of the glycolate pathway that functions in photorespiration. However, only a few leaf peroxisomal proteins, that is hydroxypyruvate reductase (HPR), glycolate oxidase (GO) and alanine:glyoxylate aminotransferase 1 (AGT1), have been characterized, and other functions in leaf peroxisomes have not been solved. To better understand the functions of leaf peroxisomes, we established a method to isolate leaf peroxisomes of greening cotyledons. We analyzed 53 proteins by MALDI-TOF MS and then identified 29 proteins. Among them, five proteins are related to the glycolate pathway, four proteins function in scavenging of hydrogen peroxide and additionally 20 novel leaf peroxisomal proteins were identified. In particular, protein kinases and protein phosphatase were first identified as peroxisomal proteins suggesting that protein phosphorylation is one of the regulatory mechanisms in leaf peroxisomes. Novel leaf peroxisomal proteins contained five PTS1-like proteins that have sequences where one amino acid is substituted with another one in PTS1 sequences. The PTS1 motif was suggested to have novel PTS1 sequences.     

Peroxisomes in molluscs, characterization by subcellular fractionation combined with western blotting, immunohistochemistry, and immunocytochemistry

Peroxisomes of the digestive glands of mussels, Mytilus galloprovincialis Lmk, were investigated by immunoblotting and immunohistochemistry using rabbit antibodies against several mammalian hepatic peroxisomal proteins. Western blot analysis of main subcellular fractions revealed immunoreactive polypeptides with molecular weights comparable to those of the corresponding mammalian hepatic proteins. They could be localized to the peroxisomal matrix in the case of catalase, multifunctional enzyme (PH), and palmitoyl-CoA oxidase (AOX), and to the peroxisomal membrane in respect to PMP 70. The purification of peroxisomes by metrizamide density gradient centrifugation revealed the existence of two subpopulations with densities of 1.16 and 1.20 g cm^–3 exhibiting different protein compositions. In paraffin sections, positive immunolabeling for catalase was distributed along the apical cytoplasm of the epithelia of digestive ducts and stomach and throughout the cytoplasm of digestive tubule cells. The peroxisomal β-oxidation enzymes, AOX and PH, also appeared predominantly in the ducts and the stomach epithelia with a weaker immunolabeling in the tubules. At the electron microscopic level a clear labeling with gold particles was observed in the peroxisomal matrix with the anti-guinea pig catalase antibody. In addition to peroxisomes, the anti-PH antibody also labeled the mitochondria. The similarity in the protein composition of molluscan and mammalian peroxisomes as revealed by the present study indicates that those proteins have been well conserved in evolution suggesting that functionally peroxisomes in molluscs could also be involved in the metabolism of lipids and in detoxification of xenobiotics. Thus, the antibodies tested could provide useful tools for detection of peroxisomal induction in molluscan biomonitoring programs for the assessment of aquatic environmental pollution. Accepted: 19 October 1999