Letters to the Editor pubs.acs.org/crt
Letter to the Editor Regarding the Article by Natsch et al., 2015
T
hydroperoxide. In the paper by Karlsson et al., it was clearly demonstrated that ketoprofen generates major amounts of the tryptophan-lysine adduct after only a few minutes, whereas 30 min to 1 h of UV-irradiation was needed to obtain similar levels in the absence of ketoprofen.2 In the current paper by Natsch et al., no such comparison is made. To establish if hydroperoxides give significantly higher amounts of formylkynurenine than natural sunlight, a negative control experiment must be performed. 2. When describing the background, the authors state the following: “Our recent analytical studies examining aged perfumes retrieved from consumers could for the first time identify trace levels of the hydroperoxides in consumer products, but these levels are 3−4 orders of magnitude below the levels used in patch testing and 3−4 orders of magnitude below the inducing concentration in animal tests. Therefore, we concluded that these low levels do not represent a likely source of exposure sufficient to induce skin sensitization in a large fraction of dermatological patients.” The authors are skipping important steps and omitting published material in the research area, which makes the text misleading. The patch test concentration in the clinical studies (6% oxidized linalool containing 1% linalool hydroperoxides) is higher than the expected elicitation levels, which is in line with clinical experience for patch test preparations in general. The following results and the references are not given in the article. A clinical repeated open application test (ROAT) using several concentrations of oxidized linalool is not cited. A ROAT for oxidized linalool was performed in individuals with positive patch test reactions to oxidized linalool. Contact allergic reactions were seen to a 0.3% concentration of oxidized linalool with a known content of 560 μg/g linalool hydroperoxides when applying cream or “perfume” twice daily for up to 3 weeks.19 The concentrations used in the ROAT study were in accordance with concentrations of fragrance terpenes expected in common consumer products.20 Concentrations of linalool hydroperoxides >100 μg/g were detected in the perfumes recalled from consumers.21 Thus, comparing these results the difference in concentration of linalool hydroperoxides found in perfumes is only about 5 times lower than the levels that cause elicitation in the ROAT. It is important to be aware of that in the multicenter patch test studies referenced by Natsch et al., the hydroperoxides (the primary oxidation products) are present as ingredients of the total oxidation mixture after autoxidation of limonene or linalool.22−25 Thus, also secondary oxidation products with allergenic activity can play a role. This is not discussed in the actual article, but the reader gets the impression that the hydroperoxides are tested as single substances. Moreover, metabolic activity may take place, as shown for linalool.26 Thus, the model used does not reflect the full clinical exposure. 3. The authors make a statement on cross-reactivity, without giving the information available from earlier studies. They state
o the Editor: We would like to focus your attention on major concerns that we have regarding the article by Natsch et al. since we find that crucial information is missing making it potentially misleading.1 The article argues that oxidative events triggered by endogenous hydroperoxides and hydroperoxides/oxidants derived from xenobiotics may lead to a sensitized state detected by patch testing with high concentrations of hydroperoxides in dermatitis patients. The experiments are conducted using tryptophan without negative controls, and based on this, the results of the clinical studies are questioned. 1. The authors have applied a hypothesis suggested by Karlsson et al. on a completely different scenario.2 The study by Karlsson et al. was initiated due to numeral clinical reports suggesting that ketoprofen induces photocross-reactivity to different compounds with various structures.3−9 Further, an animal study shows that photoinduction with ketoprofen leads to photoreactions to benzophenone and tiaprofenic acid.10 In the case of linalool and limonene hydroperoxides, the situation is the opposite. No evidence of unspecific reactivity or crossreactivity has been observed.11−13 This is further discussed below. The absence of cross-reactivity in clinical studies is, in our view, very important to consider when evaluating the clinical implications of a chemical model. In the study by Karlsson et al. the researchers performed photoincubations with ketoprofen and several different amino acid analogues (cysteine, histidine, lysine, tryptophan, and tyrosine) believed to be involved in the formation of immunogenic hapten−protein complexes.2 The authors found no ketoprofen adducts, i.e., no binding of ketoprofen to any of the amino acid analogues, but large amounts of a tryptophanlysine adduct. In the current article by Natsch et al., incubations were only performed with tryptophan. For the purpose of determining whether peroxides can cause protein modification analogous to ketoprofen, it was not necessary to incubate with amino acids other than tryptophan. However, we consider that incubations must be performed with other amino acids as well or with peptides/proteins to get a relevant picture of which protein modifications to which limonene hydroperoxides and linalool hydroperoxides can lead. Such studies with limonene hydroperoxide are described in the literature.14−17 In those studies, limonene hydroperoxide was assessed toward amino acids such as alanine, cysteine, histidine, leucine, lysine, tyrosine, and tryptophan. All studies showed specific adduct formation with cysteine or histidine and the limonene hydroperoxide residue carvone. Thus, these studies indicate that hydroperoxides cause contact allergy by forming specific immunogenic hapten−protein complexes, which is in correlation with the clinical data. None of these studies are discussed or referred to, and no references are given by Natsch et al.1 Moreover, it is well known that natural sunlight causes oxidation of tryptophan.18 According to Natsch et al., only 5% of tryptophan (compound 4) is transformed to formylkynurenine (compound 5) after incubation at 36 °C for 24 h with heme (iron) and 10-fold excess of limonene or linalool © 2015 American Chemical Society
Published: October 23, 2015 2079
DOI: 10.1021/acs.chemrestox.5b00306 Chem. Res. Toxicol. 2015, 28, 2079−2081
Chemical Research in Toxicology
Letters to the Editor
the following: “Careful dose−response curves with 1 and 2 in positively-tested patients and in parallel testing of the same patients with other hydroperoxides such as, but not limited to, 3 would be a first step to understand possible cross-reactions between hydroperoxides. This should go along with evaluation of data for cross-reactions at the individual level in published studies.” The following results and the references are published and available but omitted in the article by Natsch et al. Regarding concomitant reactions to allergens where the main haptens are hydroperoxides, cross-reactivity based on structural similarity or unspecific reactions has been investigated both in experimental and in clinical studies. In experimental studies in guinea pigs, cross-reactivity was only obtained between the hydroperoxides with very similar overall structures (in the study: cumene hydroperoxide and cyclohexene hydroperoxide), while no patterns of unspecific reactions or cross-reactions were seen between hydroperoxides with different overall structures (in the study: cumene,/ cyclohexene, limonene-2-hydroperoxide, and 15-hydroperoxyabietic acid).11 In a study on colophonium-allergic patients, the main allergen in colophonium, a hydroperoxide (15-hydroperoxyabietic acid), as well as limonene-2-hydroperoxide, and linalool hydroperoxides, were tested simultaneously. Only 1 of 29 patients reacted to more than one hydroperoxide, and no evidence of unspecific reactions or cross-reactivity was observed.11 Furthermore, in a small clinical study the two main hydroperoxide analogues in oxidized limonene, limonene1-hydroperoxide and limonene-2-hydroperoxide, were tested simultaneously in 7 patients with known contact allergy to oxidized R-limonene.12 One analogue, limonene-1-hydroperoxide, although found in a lesser amount in the oxidation mixture of R-limonene, gave more positive reactions and was also shown to be a stronger allergen in the local lymph node assay (LLNA) in mice. No unspecific reactivity and no evidence of cross-reactivity were found. The result was supported when 763 consecutive dermatitis patients were tested simultaneously with limonene-1-hydroperoxide and limonene-2-hydroperoxide. Limonene-1-hydroperoxide gave more reactions in the tested patients; a few patients reacted to both analogues, and specific reactivity was recorded.13 Taken together, these studies show that cross-reactivity for hydroperoxides follow the same pattern as that seen for cross-reactivity among other haptens, i.e., that close similarity in the overall structure is required. None of these studies is discussed or referred to, and no references are given by Natsch et al.1 The text gives the impression that the unspecific reactivity caused by very high patch test concentrations is the most important explanation behind positive patch test reactions to fragrance hydroperoxides. 4. In Figure 1, the authors give a plot of patch test reactions in different test centers and find a linear relationship. The comment to the graph reads: “Thus, it is striking that a plot of published frequency of positive and doubtful reactions to 1 and 2 reveals a high correlation at the population level...” The data shown are neither explained in the text nor in the figure legend. After studying Figure 1 from different aspects, we conclude that it is most likely a graphed summation of positive and doubtful reactions for oxidized linalool and oxidized limonene, respectively, at the different test centers, according to literature data, that is displayed.24,25 As stated above, the positive and doubtful reactions in the original papers are caused by the total mixtures of oxidized linalool and limonene,
although the hydroperoxides are considered to be the main sensitizers. Another problem is the claim that the numbers given are on a population level. The numbers given in the original papers are from patch testing of consecutive dermatitis patients at the centers in the study, in all 2900 patients. Besides the incorrectness of the data, Figure 1 is not an established way to discuss results in the patch test community, and no similar graphs with this summation have to our knowledge been made, which makes this graph very difficult to interpret or compare. That the authors find the result of Figure 1 striking is surprising, as they have not proved anything. If the authors want to show that more reactions to one fragrance correlate to more reactions to another fragrance, this is not exceptional for oxidized linalool and oxidized limonene since it is well known that fragrance allergic patients often react to many fragrance allergens.27 This has been attributed to multiple exposures when using fragranced products.27 In conclusion, there are serious issues with the article that we hope the Editor will take into consideration.
Ann-Therese Karlberg*,† Anna Börje† Jean-Pierre Lepoittevin‡ Elena Giménez-Arnau‡ Johanna Brar̊ ed Christensson†,§ Lina Hagvall§ †
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Department of Chemistry and Molecular Biology, Dermatochemistry and Skin Allergy, University of Gothenburg, SE-412 96 Gothenburg, Sweden ‡ Laboratoire de Dermatochimie, ILB 4, rue Blaise Pascal CS 90032, F-67081 Strasbourg cedex, France § Department of Dermatology, Sahlgrenska Academy, University of Gothenburg, SE-405 03 Gothenburg, Sweden
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
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REFERENCES
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