Organohalogens Naturally Biosynthesized in Marine Environments

Apr 4, 2018 - Telephone: +1-(530)-752-6696. ... expression of AhR responsive genes and (2) ryanodine receptors (RyRs), anchored within sarcoplasmic/en...
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Ecotoxicology and Human Environmental Health

Organohalogens naturally biosynthesized in marine environments and produced as disinfection by-products alter sarco/endoplasmic reticulum Ca dynamics 2+

Jing Zheng, Shaun M. K. Mckinnie, Abrahim El Gamal, Wei Feng, Yao Dong, Vinayak Agarwal, William Fenical, Abdhesh Kumar, Zhengyu Cao, Bradley S. Moore, and Isaac N Pessah Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00512 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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C Ryanodine

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C Compound

Activation Phase

Inhibition Phase

EC50 (µM) (95% CI)

Efficacy (Fold of Veh) (95% CI)

IC50(µM) (95% CI)

0.36

3.8

3.0

(0.27-0.87) 0.60 (0.51-1.0)

(3.3-5.8) 3.01 (2.6-4.3)

(2.4-3.7) 4.9 (4.5-5.5)

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24.5 (17.9-42.2)

10.3 (9.0-12.7)

83.0 (82.1-84.1)

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38.6 (23.1-114.2)

9.1 (7.4-13.7)

~121.6 (N.D)

6 28

(N.D.) Not defined

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Arsenazo III Ca2+

RyR1

A b 65 s or 0 - ba 70 n 0n c e m

Absorbance

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l na i m e Lu Sid

Le ak

SERCA 1a

Cytoplasmic Side

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Organohalogens naturally biosynthesized in marine environments and produced as

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disinfection by-products alter sarco/endoplasmic reticulum Ca2+ dynamics

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Jing Zheng

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Vinayak Agarwal 3#, William Fenical 3, Abdhesh Kumar 3, Zhengyu Cao 1,2, Bradley S. Moore 3

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and Isaac N. Pessah 1*

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1

California, Davis, USA 2

Department of TCM Pharmacology, School of Traditional Chinese Medicines, China

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Pharmaceutical University, Nanjing, China 3

Center for Oceans and Human Health, Scripps Institution of Oceanography & Skaggs School

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, Shaun M. K. McKinnie 3, Abrahim El Gamal 3, Wei Feng 1, Yao Dong 1,

Department of Molecular Biosciences, School of Veterinary Medicine, University of

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1,2

of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, USA 3#

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current location: Parker H. Petit Institute for Bioengineering & Bioscience, Georgia Institute of Technology, Atlanta, USA

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*

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+1-(530)-752-6696.

To whom correspondence may be addressed. Email: [email protected]. Telephone:

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ABSTRACT

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Contemporary sources of organohalogens produced as disinfection by-products (DBPs) are

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receiving considerable attention as emerging pollutants because of their abundance,

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persistence, and potential to structurally mimic natural organohalogens produced by bacteria

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that serve signaling and/or toxicological functions in marine environments. Here we tested 34

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organohalogens from anthropogenic and marine sources to identify compounds active

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towards ryanodine receptor (RyR1), known toxicological targets of non-dioxin-like

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polychlorinated

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[3H]Ryanodine

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organohalogens. Further analysis indicated that 2,3-dibromoindole (14), tetrabromopyrrole

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(31) and 2,3,5-tribromopyrrole (34) at 10µM were the most efficacious at enhancing [3H]Ry

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binding. Interestingly, these congeners also inhibited microsomal sarcoplasmic/endoplasmic

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reticulum (SR / ER) Ca2+ ATPase (SERCA1a). Dual SERCA1a inhibition and RyR1 activation

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triggered Ca2+ efflux from microsomal vesicles with initial rates rank ordered 31>34>14.

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Hexabromobipyrroles (25) enhanced [3H]Ry binding moderately with strong SERCA1a

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inhibition,

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ethyl-4-bromopyrrole-2-carboxylate (27) were inactive. Of three PBDE derivatives of marine

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origin active in the [3H]Ry assay, 4’-hydroxy-2,3’,4,5’,6-pentabromodiphenyl ether (18) was

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also a highly potent SERCA1a inhibitor. Molecular targets of marine organohalogens that are

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also DBPs of emerging environmental concern are likely to contribute to their toxicity.

biphenyls ([3H]Ry)

whereas

(PCBs) binding

pyrrole

and

polybrominated

screening

(24),

(1.5-fold at 2µM when compared to baseline (binding measured in the

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presence of Veh alone, p<0.05 vs. Veh, Figure 1). These and an additional 4 HOCs (15, 24,

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26 and 27) were selected for further study based on their structural similarities to establish an

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initial structure activity relationship (SAR) using [3H]Ry, Ca2+ ATPase, and Ca2+ flux

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measurements. Based on structural homologies, these compounds were classified into two

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major categories: (A) brominated pyrroles, bipyrroles, maleimides and indoles, and (B)

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PBDE derivatives, as shown in Figure 2.

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Active HOCs within Group A enhanced the binding of [3H]Ry to RyR1 in a

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concentration-dependent manner and their relative efficacies at 10 µM ranked 14>31>13=34,

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whereas 24, 26 and 27 were inactive (Figure 3A, B). Interestingly 6 (2,3-dibromomaleimide),

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a naturally produced marine HOC, showed highly potent biphasic influences on RyR1,

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stabilizing the open state of RyR1 with an EC50 of 0.36 µM, and inhibiting with an IC50 of 3.0

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µM (Figure 3A, C; Table insert). Compound 6 was shown to form Michael adducts with

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protein Cys 47, and therefore raised the question whether its biphasic actions results from

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forming covalent adducts to hyper-reactive RyR1 Cys residues previously identified to confer

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redox-sensing properties and regulation to the RyR1 complex 48-52. To better define the

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possible role of Cys-adducts in the biphasic response observed with 6, the

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concentration-effect relationship was extended to determine whether 31 and 34, which are

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unable to form Cys-adducts, and commercially available 2, 3-dibromo-N-methylmaleimide

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(28) was tested. Indeed, all three compounds produced biphasic modulation of RyR1 (Figure

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3C), indicating that Michael addition to one or more reactive Cys was not required for the

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biphasic response. This conclusion was further supported by previous data indicating that

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4'-hydroxy-2,2',4,5'- tetrabromodiphenyl ether (4-OH-BDE-49), also incapable of forming

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Michael adducts also exhibits biphasic actions toward RyR1, whereas BDE-49 activated in a

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monotonic manner 53. Thus, biphasic activity was unlikely to be mediated by Cys

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modifications on RyR1, but more likely mediated by interaction with multiple binding sites

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on the homotetramer. 6-OH N-methylation of 6 decreased the apparent activation potency by

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1.7-fold (p80% inhibition at 2.0 µM (Figure 5D and F), and ~90% inhibition at 10 µM (Figure

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7). At 10 µM compounds 31, 14, 6, and 34 showed intermediate SERCA1a inhibition (80, 60,

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50, and 48%, respectively) (Figure 5B, 5E, Figure SI 1 and 5C), whereas 24, 26 and 27,

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which were inactive towards RyR1, were also inactive towards SERCA1a (Figure 5G).

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The degree and distribution of pyrrole bromination was an important factor for both RyR1

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and SERCA1a activities, with 31 (tetrabromination) being most active, 34 being intermediate

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(2, 3, 5-bromination), and 26 (2, 3, 4-bromination) being inactive in both assays. These data

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indicate diortho-bromine substitution is an important contributor for both RyR1 and

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SERCA1a activity. This also extended to compound 25 (hexabromobipyrroles) that has two

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ortho-bromine substituents and possesses similar RyR1 activity as compound 34 (Figure 1).

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Compound 25 also exhibited the strongest SERCA inhibition (Figure 5D). As shown in the

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Figure SI 3, compound 25 diminished the loading capacity of calcium into the microsomal

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vesicles in a concentration-dependent manner, an effect compounded by targeting both RyR1

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activation and SERCA1a inhibition.

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Interestingly, 0.6-1.0 µM compound 6, a dibromomaleimide maximally activated RyR1

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(Figure 3) without inhibition of SERCA1a (Figure SI 1). In fact, the RyR1 inhibitory phase

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of 6 was detected at 18>34>25>14. The initial rates of Ca2+ efflux triggered

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by these HOCs followed their respective activities towards RyR1 and SERCA1a with the

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exception of the dibrominated indole 14 whose activity was significantly under-predicted by

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the Ca2+ flux rates it produced at either 5 or 10 µM, concentrations anticipated to largely

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stabilize a RyR1 open channel state while inhibiting SERCA1a ~55% (Figure 7). The

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discrepancy with compound 14 could be explained if an extended amount of time is needed

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to fully manifest stabilization of the open channel state, which is afforded by the 3-hour

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incubation in [3H]Ry binding assays but not the Ca2+ assay. Moreover, compound 14 may

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also inhibit RyR1 leak-states, which if blocked, can dramatically increase the Ca2+ loading

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capacity of the microsomal vesicles. Brominated macrocyclic bastadins isolated from the

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marine sponge Ianthella basta

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to influence the balance of channel and leak states of RyR1, although whether this

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mechanism explains the results obtained with compound 14 remains to be determined.

61-66

and anthropogenic PCBs

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have been previously shown

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Considering the potent biphasic activity of compound 6 on RyR1 (Figure 3) and its

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modest inhibitory activity toward SERCA1a (SI Figure 1), it was not unexpected that high

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concentrations (5 and 10 µM), concentrations expected to completely inhibit RyR1 channels,

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failed to trigger net Ca2+ efflux added to Ca2+-loaded microsomal vesicles (when the

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extravesicular free Ca2+ is very low; ~100 nM) (Figure SI 2). However, an additional a large

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bolus (90 nmol) of Ca2+ to trigger Ca2+-induced Ca2+-release resulted in significantly higher

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rates of release than control (** p<0.01, SI Figure 2A and B). The sensitizing influences on

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calcium-induced calcium release (CICR) were observed in the presence of nanomolar 6 that

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should partially activate RyR1 without any SERCA1a inhibition, although their magnitude

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did not reach statistical significance (SI Figure 2C and D).

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The present results are significant because they identify several prominent naturally

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synthesized marine HOCs that directly target two major proteins whose functions work in

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opposition to tightly regulate net ER/SR Ca2+ dynamics, and thus, are essential for shaping

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localized and global Ca2+ signals that regulate a plethora of cellular functions 16, 67. From the

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perspective of chemical ecology, tetrabromopyrrole (31) has been recently isolated from

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Pseudoalteromonas bacteria and shown to serve specific habitat cues for the attachment and

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metamorphosis of benthic marine invertebrates, including coral Acropora millepora larvae

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and multiple Caribbean corals (Orbicella franksi and Acropora palmata) that settle onto

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crustose coralline algae 29, 30, 68. The current findings may have widespread implications since

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motility, secretion and metamorphosis of planula are associated with the creation of

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crystalline substratum calcification mediated by precise Ca2+ signals

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tetrabromopyrrole and hexabromobipyrroles, extracted from Pseudoalteromonas bacteria that

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often associate with coralline algae-associated bacterium and Chromobacterium were

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reported to possess a spectrum of antibiotic, antifungal, antiprotozoal, antipathogen and

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antineoplastic activities69-72, 73. Recently, natural sources of HOCs were shown to accumulate

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. HOCs, including

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in marine organisms, especially apex predators such as seabirds and dolphins 74-77.

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A second, equally significant implication is the recent detection of several halopyrroles

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in DBPs, including compounds 31 and 34, found in waste and drinking water treatment

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streams are cytotoxic, genotoxic and carcinogenic to mammalian cells31, 32, 34. The newly

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identified targets reported here and their neuroactive properties reported in the following

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paper (in preparation) provide important mechanistic clues about their potential disruptive

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influences on marine ecology and contributions to human health risks, especially given recent

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evidence that HOCs of biosynthetic and anthropogenic origins accumulate in marine

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mammals and human breast milk 24, 27, 74, 75, 77, 78.

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Figure Legends

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Figure 1. Screening results of 34 organohalogens using [3H]Ry binding analysis.

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Compounds in red color represent significant effects toward RyR1 were found at 2µM at a

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threshold of p<0.05 compared with vehicle control using one-way ANOVA followed by

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Dunnett's multiple comparisons test. Compounds in green color were those inactive toward

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RyR1, and they are selected because of their structure properties. Data shown in the figure

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were conducted from 2-3 different preparations with triplicates for each preparation and were

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normalized to 1% DMSO control (% Veh) and expressed as Mean ± SD. Table SI 1

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summarizes the chemical structures tested.

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Figure 2. Compounds selected for further evaluation for their activities toward RyR1,

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SERCA1a, and microsomal Ca2+ flux. (A) Structures of selected pyrroles, bipyrroles and

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indoles. (B) Structures of PBDE derivatives of marine selected for further analysis in [3H]Ry

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binding, SERCA1 activity and Ca2+ assays. (C) Structure of ryanodine.

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Figure

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structure-activity relationship toward RyR1. Panel A shows concentration-response

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curves expressed as specifically bound [3H]Ry to RyR1-enriched microsomal membranes

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(JSR) isolated as described in METHODS. Data were fit with three-parameter or Bell-shaped

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(compound 6) non-linear regression with Graph Pad 7.03. Panel B shows stimulation relative

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to vehicle (1% DMSO) control (% of Veh; dashed line). C shows results from an expanded

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concentration range to quantify biphasic parameters fitted as described in Panel A for

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compounds 6, 28 and 31. The activation phase for 34 was fit with a three-parameter equation

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but lacked sufficient data to fit the inhibition phase (dashed line). Parameters obtained from

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curve-fitting are summarized in inserted table. Data shown are from 2-3 independent

3.

Brominated

pyrroles,

bipyrroles

and

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exhibit

stringent

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preparations conducted in triplicates or quintuplicates and expressed as Mean ± SD. With

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Graph Pad 7.03, one-way ANOVA followed by Tukey’s multiple comparisons test was used

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to determine the significance among specific compounds. * p