Cell-, tissue-, sex- and developmental stage-specific expression of mouse flavin-containing monooxygenases (Fmos)

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Abstract

The cell-, tissue-, sex- and developmental stage-specific expression profiles of five members of the flavin-containing monooxygenase (FMO) family, FMO1, 2, 3, 4 and 5, were investigated in 129/SV mice, using isoform-specific antisense RNA probes. In situ hybridization localized FMO1 and 5 mRNAs to the perivenous, and FMO 2, 3 and 4 mRNAs to the periportal, regions of the liver. In kidney, each FMO mRNA is localized to the distal and proximal tubules and collecting ducts; FMO1 mRNA is present also in the glomerulus. In lung, FMO1 and 3 mRNAs are expressed in the terminal bronchiole, and FMO1 mRNA also in the alveoli. FMO1 mRNA is present in neurons of the cerebrum and in the choroid plexus. RNase protection assays showed that the most abundant isoform in newborn liver, lung, kidney and brain, and in adult lung and kidney is FMO1, but in adult liver FMO5 is present in greatest amounts. In liver, lung and kidney, expression of Fmo1, 3 and 5 peaks at 3 or 5 weeks of age, but in the brain, Fmo1 expression is greatest in newborns. In the kidney, FMO5 mRNA abundance is fourfold greater in males than in females, at all stages of development. Our results demonstrate that Fmo1, 2, 3, 4 and 5 exhibit distinct cell-, tissue-, sex- and developmental stage-specific patterns of expression.

Introduction

The flavin-containing monooxygenases (FMOs) [EC 1.14.13.8] constitute a family of proteins that play an important role in the detoxification of many foreign chemicals, including psychoactive drugs, pesticides and dietary-derived compounds [1]. Humans have five genes, named Fmo15[2], and six pseudogenes, Fmo6P11P[3]. In mice, there are nine Fmo genes, which are named Fmo16 and 9, 12 and 13[3].

FMO1 and 3 are considered to be the most important members of the FMO family with respect to the metabolism of foreign chemicals [4]. In humans, FMO1 is expressed in fetal kidney and liver and in the adult kidney, but not in adult liver [5]. This is in contrast with other mammals, such as pig [6], rabbit [7], rat [8] and mouse [9], which do express FMO1 in adult liver. The expression of FMO3 is switched on in the liver after birth in humans [5], [10] and in mice [9]. In most humans, functional FMO2 is not expressed because of nonsense and/or frameshift mutations in the FMO2 gene [11], [12], [13]. An allele encoding a full-length functional FMO2 is present at a frequency of 13% in individuals of African descent [14]. In rabbit [15], mouse [16] and guinea pig [17], a full-length functional FMO2 is expressed in the lung. But the FMO2 gene of Rattus norvegicus encodes a non-functional protein [18]. Current evidence indicates that FMO6, in humans, also does not code for a functional protein [13], [19]. FMO5 is expressed in the liver of humans [20], rabbit [21], guinea pig [20] and mouse [22] and FMO4 mRNA has been detected in low amounts in several human tissues [23], [24]. Analysis of mouse sequence databases revealed no cDNAs for FMO12 and 13 and just one, isolated from a 0-day neonate mouse head library, for FMO9 [3]. Thus, none of these three genes appears to be significantly expressed in the adult mouse.

Members of the family exhibit marked developmental stage- and tissue-specific patterns of expression, which differ among species [3], [5]. Differences in the patterns of expression of FMOs have implications for the ability of an organism to respond to substrates of these enzymes that are present in its environment and diet, or that are used as therapeutic drugs in clinical or veterinary medicine.

Hormonal regulation appears to play a key role in some of the species- and gender-specific differences observed in FMO expression. For example, in mice Fmo3 is down-regulated in adult male liver by testosterone [25], whereas male rats have a higher hepatic FMO-related enzyme activity than do females [26]. In vitro studies, using co-cultures of male rat hepatocytes, showed that 17 beta-estradiol [27] and thyroid hormones [28] down-regulate the expression of FMO. In humans the symptoms of a fish-like body odour, associated with the inherited disorder trimethylaminuria (fish-odour syndrome), are exacerbated in some females during menstruation [29], indicating a hormonally mediated decrease in the abundance of FMO3, whose gene, when mutated, gives rise to the disorder [30].

The mouse is increasingly being used as a model organism as researchers seek to understand the function of mammalian genes and the role of specific proteins in health and disease. We have investigated the expression profiles, in the mouse, of five members of the mammalian FMO family, FMO1, 2, 3, 4 and 5. To define these profiles precisely, we have used both in situ hybridization analysis and quantitative RNase protection assays with mouse FMO isoform-specific antisense RNA probes. This allows a comparison of the abundance and cellular location of each isoform within a single tissue, among tissues, and at different stages of development. The quantitative nature of RNase protection assays means that direct comparisons can be made between data derived from the mouse and published data obtained from human tissues.

Section snippets

Animals

Animals were purchased from Harlan and maintained on the Harlan Teklad TRM Rat/Mouse diet. They had free access to food and water and were sacrificed by cervical dislocation.

Sexing of newborn

The sex of newborn 129/SV mice was determined by PCR-amplification of a Y chromosome-specific sequence, from tail DNA, using the primer pair: forward, YMTFP1, 5′-ctggagctctacagtgatga-3′; reverse, YMTRP1, 5′-cagttaccaatcaacacatcac-3′ [31]. Tail DNA was isolated as described [32]. As a positive control, a sequence of the

Results and discussion

We have used in situ hybridization to determine, and compare, the cell type-specific expression of Fmo1, 2, 3, 4 and 5 in liver, lung, kidney and brain of the adult 129/SV mouse. These experiments show that the regional localization in the liver lobule of FMO1, 2, 3, 4 and 5 mRNAs is not the same. FMO1 and 5 mRNAs are detected across the acinus, with a concentration gradient decreasing from the perivenous to periportal region (Fig. 1a and h). In contrast, expression of FMO2 and 4 is restricted

Acknowledgements

We thank Dr. Anoop Kumar, Department of Biochemistry and Molecular Biology, University College London, for help with tissue preparation and photography. The work was supported by a grant from the Wellcome Trust (No. 053590).

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