Sn-protoporphyrin inhibits both heme degradation and hemozoin formation in Rhodnius prolixus midgut C.D. Caiaffa a , R. Stiebler a , M.F. Oliveira a , F.A. Lara b , G.O. Paiva-Silva a, c , P.L. Oliveira a, c, * a Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, CEP 21941-590, Rio de Janeiro, Brazil b Laboratório de Microbiologia Celular, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil c Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Brazil article info Article history: Received 6 June 2010 Accepted 18 August 2010 Keywords: Heme Heme oxygenase Hematophagy Rhodnius prolixus Oxidative stress abstract Hematophagy is a feeding habit that involves the ingestion of huge amounts of heme. The hematoph- agous hemipteran Rhodnius prolixus evolved many genetic resources to protect cells against heme toxicity. The primary barrier against the deleterious effects of heme is the aggregation of heme into hemozoin in the midgut lumen. Hemozoin formation is followed by the enzymatic degradation of heme by means of a unique pathway whose end product is dicysteinyl-biliverdin IX-g (Rhodnius prolixus biliverdin, RpBv). These mechanisms are complemented by a heme-binding protein (RHBP) in the hemolymph that attenuates the pro-oxidant effects of heme. In this work, we show that when insects are fed with blood enriched with a heme analog, Sn-protoporphyrin (SnPP-IX), both hemozoin synthesis and RpBv production are inhibited in a dose-dependent manner. These effects are accompanied by increased oxidative damage to the midgut epithelium and inhibition of oviposition, indicating that hemozoin formation and heme degradation are protective mechanisms that work together and contributed to the adaptation of this insect to successfully feed on vertebrate blood. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Hematophagy arose independently in many insect orders, and during the course of evolution, different groups of blood-feeding insects acquired specific ways to manage and digest blood (Lehane, 1991; Mans and Neitz, 2004). Hematophagous insects can ingest in a single meal a huge amount of vertebrate blood, which contains about 10 mM of heme bound to hemoglobin. Heme is a potentially harmful molecule, exerting its toxicity either by promoting the formation of reactive oxygen species or alternatively, by insertion of free heme into phospholipid membranes eventually leading to cellular lysis (Kumar and Bandyopadhyay, 2005; Ryter and Tyrrell, 2000; Schmitt et al., 1993; Tappel, 1955; Wijayanti et al., 2004). The metabolic adaptations required for a blood diet should maintain cellular homeostasis in a pro-oxidant environment created by an increased heme concentration (Graca-Souza et al., 2006). One of the preventive mechanisms of Rhodnius prolixus against free heme injury is to promote the formation of a crystalline aggregate called hemozoin (Oliveira et al., 1999), a protective mechanism also found in the malaria parasite, Plasmodium falciparum (Slater et al., 1991), and in the blood fluke, Schistosoma mansoni (Oliveira et al., 2000a). In R. prolixus, this type of defense can account for most of the heme found in the midgut lumen and therefore it has been suggested that it has a protective role (Oliveira et al., 2000b). However, a need for other complementary mechanisms, such as antioxidant enzymes, has already been pointed (Paes et al., 2001). Another important defense mechanism against heme toxicity is achieved through the presence of heme-binding proteins in the hemolymph of R. prolixus. The Rhodnius heme-binding protein (RHBP) is a monomeric heme- binding protein in the range of 12 KDa that binds heme as a prosthetic group (Oliveira et al., 1995). RHBP controls free heme reactivity and acts as a transporter of heme to the ovaries (Dansa-Petretski et al., 1995; Machado et al., 1998). From bacteria to men, heme disposal is accomplished by the action of heme oxygenase, an enzyme that oxidatively cleaves the porphyrin ring of the heme, releasing iron, carbon monoxide and biliverdins (Montellano, 2000; Tenhunen et al., 1969; Wilks, 2002). Therefore, it is conceivable that enzymatic degradation by the microsomal heme oxygenase should be a key aspect of heme homeostasis and an important defense against heme toxicity in blood-feeding insects. Sir Vincent Wigglesworth, in a pioneer study (Wigglesworth, 1943), described the presence of green pigments in * Corresponding author. Universidade Federal do Rio de Janeiro, Prédio do CCS/Bl. D, subsolo, sala DS-05, Cid. Universitária e ilha do Fundão, Rio de Janeiro, RJ 21941- 590, Brazil. Tel.: þ55 21 2562 6751; fax: þ55 21 2270 8647. E-mail address: pedro@bioqmed.ufrj.br (P.L. Oliveira). Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb 0965-1748/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2010.08.005 Insect Biochemistry and Molecular Biology 40 (2010) 855e860