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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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Environmental Science & Technology

ACS Paragon Plus Environment


Figure 1


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A

24

6

B

26

34

31

13

28

14

15

18

17

19

C Ryanodine

Figure 2


27

25

A

B


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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)

31

24.5 (17.9-42.2)

10.3 (9.0-12.7)

83.0 (82.1-84.1)

34

38.6 (23.1-114.2)

9.1 (7.4-13.7)

~121.6 (N.D)

6 28

(N.D.) Not defined

Figure 3


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A

Figure 4

B



A

D

Figure 5

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C

B

E

F


G

A

B


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

Figure 6

C

D

E

F

G

H



Figure 7


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1

Organohalogens naturally biosynthesized in marine environments and produced as

2

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

3 4

Jing Zheng

5

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

6

and Isaac N. Pessah 1*

7

1

California, Davis, USA 2

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

10 11

Pharmaceutical University, Nanjing, China 3

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

12 13

, 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

8 9

1,2

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

14

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

15 16 17 18

*

19

+1-(530)-752-6696.

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



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20

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,

23

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

24

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

25

organohalogens from anthropogenic and marine sources to identify compounds active

26

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

27

polychlorinated

28

[3H]Ryanodine

29

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

30

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

31

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

32

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

33

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

34

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

35

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

38

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

39

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

155

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

156

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

157

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

158

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

159

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

160

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),

164

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

228

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

229

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

16

have been previously shown

282

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

283

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

284

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


29

. 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

318 319

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

325

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

326

summarizes the chemical structures tested.

327 328

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

331

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

332 333

Figure

334

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

336

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

337

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

338

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

339

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

340

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

341

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

342

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

3.

Brominated

pyrroles,

bipyrroles

and


indoles

exhibit

stringent

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343

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

344

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

345

to determine the significance among specific compounds. * p

Organohalogens naturally biosynthesized in ... - ACS Publications

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