HIV antiretrovirals in the environment

H Bouwman, B Vogt, D van Aswegen, D Fouché, E Gerber, L van der Merwe, C Bezuidenhout, L Bothma, R Muller, S Horn, T Vogt, P Bester, R Pieters

HIV antiretrovirals in the environment

H Bouwman, B Vogt, D van Aswegen, D Fouché, E Gerber, L van der Merwe, C Bezuidenhout, L Bothma, R Muller, S Horn, T Vogt, P Bester, R Pieters

Ecotoxicologists

HIV antiretrovirals in the environment

H Bouwman, B Vogt, D van Aswegen, D Fouché, E Gerber, L van der Merwe, C Bezuidenhout, L Bothma, R Muller, S Horn, T Vogt, P Bester, R Pieters

Microbiologists

HIV antiretrovirals in the environment

H Bouwman, B Vogt, D van Aswegen, D Fouché, E Gerber, L van der Merwe, C Bezuidenhout, L Bothma, R Muller, S Horn, T Vogt, P Bester, R Pieters

AUTHeR

AUTHeR

• Africa Unit for Transdisciplinary Health Research

• Focused on bio-psycho-social health in the context of epidemiology toward preventing illness, maintaining and promoting health and facilitating quality health systems.

• http://www.nwu.ac.za/health-sciences/auther

INTRODUCTIONRecommendations of previous ARV project:

INTRODUCTIONAims of current project

• Follow and predict changes in HIV-ARV use patterns

• Improve the sampling, extraction, and analytical procedures

• Give attention to potential stable breakdown products

• Determine spatial and temporal patterns of ARVs in waste water, treated effluent, natural water, and drinking water

• Determine the effectiveness of ARV removal via WWTPs

• Determine effects in the environment

• Conduct a risk assessment

INTRODUCTION

• Pharmaceuticals and personal care products 3000+ detected in environment; drinking water too

INTRODUCTION

• Pharmaceuticals and personal care products 3000+ detected in environment;

• Eg. anti-inflammatories, analgesics, anti-pyretics, anti-epileptics, anti-depressants, beta-blockers, antibiotics,

drinking water too

ANTIVIRALs, etc.

INTRODUCTION

• Used therapeutically: have biological activity

• Ineffectual waste water treatment

• Large volumes excreted, diluted• 3.4 million South Africans on ARVs (NDoH, 2016)

• SA has largest antiretroviral therapy program globally (WHO, 2017)

• Surface water and groundwater

• ARVs in SA water:

Wooding et al 2017 Journal of

Chromatography A 1496:122–132Efavirenz & nevirapine 0.16–227 ng/L

Schoeman et al 2015 J Chromatogr

Sep Tech 6: 272

Efavirenz & nevirapine 350–7100 ng/L

Wood et al 2015 Environmental

Pollution 199:235–243Mixtures: 26.5–430 ng/L (Means)

INTRODUCTION

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INTRODUCTION

• Side effects of ARVs at therapeutic levels:• Hypersensitivity eg skin rash, GI symptoms

• Mitochondrial toxicity eg hepatic steatosis, lactic acidosis, myopathy

• Metabolic & cardiovascular abnormalities eg at redistribution, atherosclerosis, glucose intolerance

• Central nervous system effects eg psychosis, headache, depression

• Bone abnormalities eg reduced bone density

• Nephropathy eg kidney stones

• Potentially teratogenetic

INTRODUCTION

• What are the effects at environmental levels?

• On organisms not supposed to be exposed?• Bacteriophages (viruses infecting bacteria)

• Freshwater snails (invertebrate)

• In vitro mammalian cell assays (endocrine disruption)

BACTERIOPHAGES

• Viruses affecting bacteria

• One class natural occurring phages = coliphages

• Coliphages infect E. coli

• Bacteria NB in waste water treatment

• Previous results (Ms Thumi Letsi):

10 µg/L 100 µg/L 1000 µg/L

Nevirapine Opaque plaques No plaques Opaque plaques

Abacavir Opaque plaques Opaque plaques No plaques

Efavirenz No plaques No plaques Clear plaques

Tenofovir No plaques No plaques Opaque plaques

Nelfinavir Opaque plaques Opaque plaques Opaque plaques

BACTERIOPHAGES

• How do ARVs influence naturally occurring viruses?

• Culturing coliphages, exposing them to ARVs, then testing their ability to kill E. coli

• 2 classes coliphages: F-specific RNA & somatic

• Determining how the ARVs may influence host expression of phage binding sites

fertility-pilli

cell wall

phage

TEM photo's by Dr. A. Jordaan & Me. L. Bothma

• Why snails?• Invertebrate integral to aquatic food web

• Bulinus tropicus member of 2nd most diverse phylum

• Rapid reproductive cycle

• Bio-indicator of pollution

• Determine effect of ARVs on:• Embryonic growth

• Embryo mortality

• Possible endocrine disruption

• Reproduction over time

FRESHWATER SNAILS

texasaquaticscience.org

Photo: D v Aswegen

FRESHWATER SNAILS

• Method

Photo: D v Aswegen

FRESHWATER SNAILS

• Preliminary results: Efavirenzlater hatching

FRESHWATER SNAILS

• Preliminary results: Lamivudine

In vitro TISSUE CULTURE ASSAYS

• Endocrine disruptive effects

• Two mechanisms of action:• reporter gene assays

• steroidogenesis assay

Reporter gene assays

Reporter gene assays

Reporter gene assays

TranslationNatural response

mRNA

Translation

mRNA

Light emission

Reporter gene assays

Steroidogenesis assay

• Results• No activation of AR

• Slight inhibition of AR

• Effects on steroidogenesis assay

TISSUE CULTURE ASSAYS

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Lamivudine concentration (ng/L)

Testosterone

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Stavudine concentration (ng/L)

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Ritonavir concentrations (ng/L)

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Lopinavir concentrations (ng/L)

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WAY FORWARD

• Generate results in the microbial study

• Complete snail exposures

• Tissue culture work:• Compare current results to that of environmental

extracts

• Compare with chemical analytical data

ACKNOWLEDGMENT

Funding: Water Research Commission

Project no: K5/2594

A study of phageA study of phageA study of phageA study of phage----host interactions in host interactions in host interactions in host interactions in

the midst of antiretroviral the midst of antiretroviral the midst of antiretroviral the midst of antiretroviral

contaminationcontaminationcontaminationcontamination

Leani Bothma, Prof. Carlos Bezuidenhout

Bacteriophages

• Bacteriophage (phage)

• Coliphage

• Somatic coliphages

• Absorb to receptors situated in cell wall.

• Escherichia coli strain C WG5 (ATCC 700078)

• Absence of DNA restriction system

• F-specific RNA coliphages

• Specifically absorb to fertility (F)-fimbriae.

• Salmonella enterica WG 49 (ATCC 700730)

• Contains transferred F-plasmid of E. coli K-12.

• Pathogenic gene has been deleted

Literature

• Somatic coliphage > F-RNA coliphage

• All faecal polluted water

• Somatic coliphage < F-RNA coliphage

• Human faecal matter

• Change in domestic water composition

• Personal care pharmaceuticals (ARV's)

Previous studies

• Me. Tumi Letsi (B.Sc Hons) 2013• Potential implications of HIV antiretrovirals on aquatic phages

A BFigure1: (A) Intact phages (image courtesy of www.scienceopen.com). (B) Damaged appendages of phages exposed to ARV's (image courtesy of Me. T. Letsi & Dr. A .Jordaan)

Phage survival in the midst of ARV pollution

• Environmental levels of ARVs will be reproduced in the laboratory

• Known phage titers will then be exposed to these levels

• Effect of ARV concentrations on phage titer will be established

• Double-agar- layer-plaque assay

• Modified ISO 10705

• Death curve

• 96 well micro plate reader

Phage-host interaction

• Do ARV's interfere with the phage-host interaction?

Fertiliti-pilli

Cell wall

Phage

A

A B

Figure 2: (A) Somatic coliphage attached to cell wall of E. coli. (B) F-RNA coliphage attached to fertiliti-pili of E. coli. TEM photo's by Dr. A. Jordaan & Me. L. Bothma (2015), NWU-Potchefstroom campus.

Establishing the genetic effects of ARV's on bacterial hosts

• Whole genome sequencing• E. coli (WG 5) & Salmonella Typhemerium enterovicar (WG 49)

• Exposure of bacterial hosts to various concentrations of ARV's active ingredients

• Nevirapine, Efavirenz, Zidovudine, Stavudine, Lamivudine, Didanosine, Lopinavir, Ritonavir

• Transcriptome analyses of differential expression of mRNA

• Quantitative analyses• ISO 10705 • 96 well micro plate reader

Double-agar-layer plaque assay ISO 10705-1 & 2

Figure 3: Photo of the double-agar-layer plaque assay being performed. The semi-

solid TYG (Tryptone Yeast Glucose extract) top-agar, containing the phage culture

and bacterial host mixture, is being poured over the solid bottom-layer TYG agar.

ssTYGA

+

Phage

+

Bacterial

host

TYGA

Plaques

A B C

Figure 4: Three different plaque morphologies observed in this study. A) Clear

plaque B) Fried egg plaque, and C) jagged edged opaque plaque