Arthropod P450 Dokuwiki

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======  Elements of a chronology of arthropod P450 research ======

The earliest studies of oxidative metabolism of insecticides in insects in the 1950's closely followed and paralleled similar studies on drug and insecticide metabolism in vertebrates. //In vivo// and intact tissue experiments were then followed by experiments with cell-free extracts and "microsomes". As details of the enzymatic requirements of such reactions in microsomal fractions emerged, it rapidly became clear that the enzyme system - initially called "[[monooxygenase|mixed function oxidase]]" and only later P450 - was essentially the same in vertebrates and insects. Both M. Agosin and L. Terriere (personal communications) related the skepticism of the (vertebrate) toxicology community in the early 1960's. Insects couldn't possibly metabolize chemicals the same way as rat liver ?   

**1958**

[[https://doi.org/10.1042/bj0700373|Fenwick]] (1958) reported the bioactivation of [[https://commonchemistry.cas.org/detail?cas_rn=152-16-9 |schradan (OMPA)]] by homogenates and a post-mitochondrial fraction from locust (//Schistocerca gregaria//) fat body. The reaction, which produced an esterase inhibitor (p-nitrophenylacetate to p-nitrophenol activity), was dependent on NADPH and aerobic conditions. This cell free system also bioactivated [[https://commonchemistry.cas.org/detail?cas_rn=115-26-4|dimefox]]. Both insecticides are phosphoroamidates, and bioactivation to the esterase inhibitors is thought to proceed by N-hydroxylation. 
Fenwick noted that the "strange need for a reducing agent in an oxidative process" had already been observed in mammalian drug metabolism (Brodie, 1956; Velick, 1956), and that the reaction mechanism might be of the type discussed by [[monooxygenase|Mason]]. 

**1960s**

Although synergists of pyrethrum had been known and used for some time, Sun and Johnson (1960) first suggested that they acted by inhibiting the oxidative metabolism of insecticides. Casida (1970) published a detailed review on the chemical diversity of synergists.


**1961**:

[[https://doi.org/10.1093/jee/54.2.340 |Agosin et al.]] (1961) described a microsomal preparation from whole adult male //Blattella germanica// that was able to convert DDT to a metabolite tentatively identified as "Kelthane" (dicofol), i.e. C1-hydroxy DDT. Highest activity was observed in the presence of NADP+, an NADPH-regenerating system, Mg++ and nicotinamide. Nearly half of the crude homogenate activity was found in the microsomal fraction, with some activity in the "nuclei" and "mitochondria" fractions. Activity was also reported for house fly and American cockroach preparations. This report followed earlier work //in vivo// that had shown conversion of DDT to kelthane in house flies (Hoskins et al, 1958, Hoskins and Witt, 1958) and Drosophila (Tsukamoto 1959,1960).

**1962**:

Arias and Terriere (1962) prepared microsomes by differential centrifugation of whole house fly homogenates (larvae, pupae or adults). These microsomes converted naphthalene by hydroxylation to 1-naphthol and 1,2-dihydro-1,2 dihydroxynaphthalene. NADPH-dependence was only observed with microsomes from older adults. 

**1964**

Morello (1964) showed that toxicity of DDT in //Triatoma infestans// was lower when DDT hydroxylation was increased by an inducer (3-methylcholanthrene) and higher when DDT hydroxylation was decreased by inhibitors (SKF 525-A and iproniazid). This was one of the first demonstration of a link between a P450 activity and in vivo toxicity.


**1965** 

Ray is usually credited with the first report of insect [[p450_spectrum|P450 spectra]] in the house fly, //Blattella germanica// and //Periplaneta americana// (J.W.Ray, Pest Infestation Research, p 59, HMSO, 1965; cited in Ray, 1967), but the first publication is that of 1967. Microsomes from whole adult house flies showed a substantial P450 peak that was converted to P420 upon addition of deoxycholate (Ray, 1967). Garfinkel (1963) had earlier reported "traces" of P450 in microsomes from lobster (//Homarus americanus//) gills (but none in hepatopancreas, green gland, gonads; one may surmise that the other tissues were consumed).


**1970s**:

Evidence of P450 induction by insecticides and other chemicals (DDT, dieldrin, phenobarbital).

Evidence of P450 multiplicity in insects, mostly from partial purification studies.

Description of developmental changes in P450 levels and P450-dependent activities in insects.

**1972**: 

Description of microsomal P450 activities in the freshwater crayfish, //Cambarus sp.// (Decapoda, Pleocyemata)(Khan et al., 1972)

**1977**:

P450 and hormones: Evidence that the conversion of ecdysone to the molting hormone 20-hydroxyecdysone is catalyzed by a P450 enzyme (Bollenbacher et al., 1977; Feyereisen 1977; Johnson and Rees, 1977).

Fluctuations in a P450 activity (benzo(a)pyrene hydroxylation) in homogenates of blue crab (//Callinectes sapidus//, Decapoda, Brachyura) green glands during a molt cycle (Singer and Lee, 1977).

**1985**:
 
Agosin (1985) and Hodgson (1985) have extensively reviewed the early literature of research on insect microsomes, microsomal oxidations of (mostly) insecticides and the emerging field of P450. These two reviews, along with that of Brooks (1979) are essential sources on the early in vivo and in vitro work.

**1987**

P450 and pheromones: Evidence that a P450 enzyme is involved in the biosynthesis of the house fly pheromone (Ahmad et al., 1987).


**1989**:

Cloning of the first insect P450, house fly CYP6A1 ([[https://doi.org/10.1073/pnas.86.5.1465 |Feyereisen et al., 1989]]). A cDNA expression library was screened with antibodies to a partially purified P450 fraction from adult house fly abdominal microsomes.

**1994**:

Functional expression of house fly CYP6A1 and P450 reductase in //E. coli// and metabolism of insecticides ([[https://doi.org/10.1021/bi00174a025|Andersen et al. 1994]])

Functional expression of //Papilio polyxenes// CYP6B1v1 and v2 in Tn5 cells by baculovirus and metabolism of plant chemicals (xanthotoxin and bergapten) ( [[https://doi.org/10.1006/abbi.1994.1175|Ma et al., 1994]] ).

Constitutive overexpression of a P450 gene (CYP6A1) controlled in //trans// (Carino et al., 1994).

Evidence that a P450 enzyme is involved in the biosynthesis of cuticular hydrocarbons (Reed et al., 1994)

**1996**:

Cloning and functional expression of house fly cytochrome b5 ([[https://doi.org/10.1074/jbc.271.43.26637|Guzov et al., 1996]])

**1998**:

Cloning and functional expression of a mitochondrial P450, house fly CYP12A1 ([[https://doi.org/10.1006/abbi.1998.0901|Guzov et al., 1998]]).

**2000-2001**:

Sequencing of the first insect genome, //Drosophila melanogaster// (Adams et al., 2000), and annotation of its CYPome ([[https://doi.org/10.1016/S0378-1119(00)00533-3|Tijet et al.,2001]])

Transgenic expression of house fly CYP6D1 in Drosophila increases microsomal benzo[a]pyrene hydroxylation (Korytko et al., 2000)

Evidence that constitutive overexpression of a P450 gene (CYP6G1) is controlled in //cis// (Daborn et al., 2001)

**2002**

Site-directed mutagenesis of CYP6B1 altered activity towards furanocoumarin substrates (Chen et al., 2002)

**2009**

RNAi of CYP6BG1 in //Plutella xylostella// was shown to increase permethrin toxicity (Bautista et al., 2009)


**2009-2011**

Sequencing of the first crustacean genome, //Daphnia pulex// (Colbourne et al., 2011), and annotation of its CYPome  ([[https://doi.org/10.1186/1471-2164-10-169|Baldwin et al., 2009]]).


**2011**:

Sequencing of the first chelicerate genome, //Tetranychus urticae// and annotation of its CYPome ([[https://doi.org/10.1038/nature10640|Grbic et al.,2011]]).

**2016-2017**

TALEN knock out of CYP9M10 in// Culex quinquefasciatus// increases permethrin toxicity (Itokawa et al.,2016)

CRISPR-Cas9 knock out of CYP6G1 in //Drosophila melanogaster// increases imidacloprid toxicity (Denecke et al., 2017)